Anti-hypertensive agents

ABSTRACT

Novel inhibitors of angiotensin converting enzyme having the general formula R--A--S--Z are disclosed as potent inhibitors of angiotensin converting enzyme and are useful anti-hypertensive agents.

BACKGROUND OF THE INVENTION

Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafterreferred to as ACE) occupies a central role in the physiology ofhypertension. The enzyme is capable of converting the decapeptideangiotensin I, having the sequence

    AspArgValTyrIleHisProPheHisLeu

to an octapeptide, angiotensin II, by removal of the carboxy-terminalHisLeu. The symbols for various chemical entities are explained in thefollowing table:

Ala=L-alanine

Arg=L-arginine

Asp=L-aspartic acid

<Glu=pyro-L-glutamic acid

Gly=glycine

Hip=Hippuric acid (Benzoyl-glycine)

His=L-histidine

Ile=L-isoleucine

Leu=L-leucine

Phe=L-phenylalanine

Pro=L-proline

ΔPro=L-3,4-dehydroproline

Ser=L-serine

Trp=L-tryptophan

Tyr=L-tyrosine

Val=L-valine

ACE=Angiotensin converting enzyme

Hepes=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

Angiotension I is formed by the action of the enzyme renin, anendopeptidase found in kidney, other tissues and plasma, acting on aserum α-2 globulin.

Blood pressure is affected by certain peptides found in the blood. Oneof these, angiotensin II, is a powerful pressor (blood pressureelevating) agent. Another, bradykinin, a nonapeptide with the sequenceArgProProGlyPheSerProPheArg is a powerful depressor (blood pressurelowering) agent. In addition to a direct pressor effect, angiotensin IIstimulates release of aldosterone which tends to elevate blood pressureby causing retention of extracellular salt and fluids. Angiotensin II isfound in measurable amount in the blood of normal humans. However, it isfound at elevated concentrations in the blood of patients with renalhypertension.

The level of ACE activity is ordinarily in excess, in both normal andhypertensive humans, of the amount needed to maintain observed levels ofangiotensin II. However, it has been found that significant bloodpressure lowering is achieved in hypertensive patients by treatment withACE inhibitors. [Gavras, I., et al., New Engl. J. Med. 291, 817 (1974)].

ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of thepenultimate peptide bond at the C-terminal end of a variety of acylatedtripeptides and larger polypeptides having an unblocked α-carboxylgroup. The action of ACE results in hydrolytic cleavage of thepenultimate peptide bond from the carboxyl-terminal end yielding asreaction products a dipeptide and a remnant.

The reactivity of the enzyme varies markedly depending on the substrate.At least one type of peptide bond, having the nitrogen supplied byproline, is not hydrolyzed at all. The apparent Michaelis constant (Km)varies from substrate to substrate over several orders of magnitude. Forgeneral discussion of the kinetic parameters of enzyme catalyzedreactions, see Lehninger, A., Biochemistry, Worth Publishers, Inc. NewYork, 1970, pp. 153-157. Many peptides which are called inhibitors ofthe enzymatic conversion of angiotensin I to angiotensin II are in factsubstrates having a lower Km than angiotensin I. Such peptides are moreproperly termed competitive substrates. Examples of competitivesubstrates include bradykinin, and the peptide BPP_(5a) (also calledSQ20475) from snake venom, whose sequence is <GluLysTrpAlaPro.

Numerous synthetic peptide derivatives have been shown to be ACEinhibitors by Ondetti, et al. in U.S. Pat. No. 3,832,337 issued Aug. 27,1974.

The role of ACE in the pathogenesis of hypertension has prompted asearch for inhibitors of the enzyme that could act as antihypertensivedrugs. See for example U.S. Pat. Nos. 3,891,616, 3,947,575, 4,052,511and 4,053,651. A highly effective inhibitor, with high biologicalactivity when orally administered, isD-3-mercapto-2-methylpropanoyl-L-proline, designated SQ14225, disclosedin U.S. Pat. No. 4,046,889 to Ondetti et al., issued Sept. 6, 1977, andin scientific articles by Cushman, D. W. et al., Biochemistry 16, 5484(1977), and by Ondetti, M. et al., Science 196, 441 (1977). Theinhibitor SQ14225 reportedly has an I₅₀ value of 2.3×10⁻⁸ M. The I₅₀value reported by Cushman, et al., supra is the concentration ofinhibitor required to produce 50% inhibition of the enzyme under astandard assay system containing substrate at a level substantiallyabove K_(m). It will be understood that I₅₀ values are directlycomparable when all potential factors affecting the reaction are keptconstant. These factors include the source of enzyme, its purity, thesubstrate used and its concentration, and the composition of the assaybuffer. All I₅₀ data reported herein have been performed with the sameassay system and same enzyme (human urinary ACE) and with anapproximately 1/2 K_(m) level of substrate and are therefore internallyconsistent. Discrepancies with data obtained by other workers may beobserved. Indeed such discrepancies do exist in the literature, forunknown reasons. See, for example, the I₅₀ values for BPP_(9a) reportedby Cushman, D. W., et al., Experientia 29, 1032 (1973) and by Dorer, F.E., et al., Biochim. Biophys. Acta 429, 220 (1976).

The mode of action of SQ14225 has been based upon a model of the activesite of ACE developed by analogy with the better known related enzyme,carboxypeptidase A. The active site was postulated to have a cationicsite for binding the carboxyl end group of the substrate and a pocket orcleft capable of binding the side chain of the C-terminal amino acid andproviding especially tight binding for the heterocyclic ring of aterminal proline residue. A similar pocket for the penultimate aminoacid residue was postulated, and the published data suggested a ratherstringent steric requirement, since the D-form of the inhibitor wassubstantially more potent than its stereoisomer or the 3-methyl andunsubstituted analogs. The sulfhydryl group on the inhibitor, postulatedto be bound at the active site near the catalytic center, was believedto play a central role in inactivation of the enzyme by combining withthe zinc moiety known to be essential for catalytic activity.Substituents on the sulfhydryl, such as a methyl group, and an S-acetylderivative, substantially reduced potency of the inhibitor. See Cushman,D. W., et al., Biochemistry, supra.

In vitro study of the mechanism by which SQ14225 and its analogs act toinhibit ACE has been somewhat hampered by the instability of thesemolecules under ambient conditions. For example, it has been observedthat a fresh aqueous solution of concentration, e.g., 1 mg per ml ofSQ14225 at a pH of about 8 becomes substantially less active uponstanding for as little as 30 minutes, and that activity continues todecrease as the solution stands for longer periods. It is believed thatthis loss in activity is mainly the result of dimerization of SQ14225occurring at the sulfhydryl end groups, whereby a disulfide is formedwhich is largely inactive as an inhibitor. Since the free sulfhydrylgroup is highly reactive and may be readily oxidized to polar acidicmoieties such as sulfone and sulfoxide groups, it may also be that theobserved in vitro loss of activity of aqueous solutions of SQ14225 onstanding is in some part a consequence of one or more such oxidationreactions, with formation of a sulfone or sulfoxide which does notfunction effectively as an inhibitor for ACE.

Such reports of SQ14225 clinical testing as are currently available,some of which refer to the compound under the name "Captopril", suggestthat the product is sufficiently stable in the normal gastric andintestinal environments of most patients to be an effective inhibitorfor ACE when administered orally. It is not yet clear, however, whetherthere may be a group of patients for which SQ14225 is substantiallyineffective. Because of the high reactivity of the free sulfhydrylgroup, SQ14225 could readily form mixed disulfides with serum, cellularproteins, peptides or other free sulfhydryl group-containing substancesin the gastric or intestinal environments, in addition to thepossibility for dimer formation or oxidative degradation reactions. Amixed disulfide with protein may be antigenic and, indeed, occasionalallergic reactions have been clinically observed. See Gavras, et al.,New England J. Med. 298, 991 (1978). Disulfides and oxidativedegradation products of SQ14225, if formed, may at best be expected tobe largely ineffective as inhibitors. It may be postulated accordinglythat dose response to SQ14225 may vary with conditions of administrationand among individual patients. Moreover, in at least some patients,unwanted side effects may occur and maintenance of an effectiveconcentration of the inhibitor in the body may be difficult to control.

Thioester compounds generally are thought to be highly reactive in thatthe thioester linkage is readily hydrolyzable to a sulfhydryl moiety anda carboxylic moiety. Thioesters are accordingly often used as activeester intermediates for acylation under mild conditions. Such groups as,e.g., acetylthio have been used as blocking groups in the above citedOndetti, et al. patents. Thioester intermediates are also postulated tooccur in the biosynthesis of cyclic peptides such as tyrocidin orgramicidin S. See Lipmann, F. in Accounts Chem. Res. 6, 361 (1973).

Thioester compounds having potent ACE inhibitory activity and oraleffectiveness as anti-hypertensive agents have been disclosed incopending applications Ser. No. 116,950, filed Jan. 30, 1980 which is acontinuation of Ser. No. 941,289, filed Sept. 11, 1978, now abandoned,Ser. No. 116,951, filed Jan. 30, 1980 which is a continuation of Ser.No. 958,180, filed Nov. 6, 1978, now abandoned, and Ser. Nos. 064,897through 064,903, all filed on Aug. 14, 1979. Ser. No. 116,951 has beenabandoned in favor of its continuation Ser. No. 295,589, filed Aug. 24,1981, which has been abandoned in favor of its continuation, Ser. No.524,204, filed Aug. 18, 1983, which has been abandoned in favor of itscontinuation, Ser. No. 680,541, filed Dec. 11, 1984, which has beenabandoned in favor of its now pending continuation, Ser. No. 850,055,filed Apr. 10, 1986. All copending applications are incorporated hereinby reference.

Compounds related to SQ14225 have been disclosed by Ondetti, et al.,U.S. Pat. Nos. 4,046,889, 4,052,511, 4,053,651, 4,113,715 and 4,154,840.Of interest are disclosed analogs of SQ14225 having the five-memberedheterocyclic ring of proline replaced by a four- or a six-membered ring.The inhibitory potencies of such analogs relative to SQ14225 are notdisclosed. Substitution of D-proline for L-proline is reported todrastically reduce inhibitory potency of 3-mercaptopropanoyl amino acids(Cushman, D. W., et al., supra).

The substitution of L-3,4-dehydroproline for proline has been studied inseveral systems. Substitution of L-3,4-ΔPro in the 7 position ofbradykinin yields a bradykinin derivative which has significantlyreduced physiological activity. See Fisher, G. H. et al., Arch. Biochem.Biophys. 189, 81 (1978). On the other hand, substitution of L-3,4-ΔProat the 3, 5, 8 or 9 position in ACE inhibitor BPP_(9a) enhances itsinhibitory activity. See Fisher, G. H. et al., FEBS Letters 107, 273(1979). In copending application Ser. No. 850,055, applicants found thatthe compounds having ΔPro, which are disclosed in said appliction, havehigh inhibitory potency and antihypertensive effectiveness. However, atpresent, no rationale can be advanced to explain the diversity ofobserved results following substitution of ΔPro for proline. Similarly,no clear picture has emerged of the effects of other proline derivativesor analogs substituted at various loci on ACE inhibitors.

To date, the effect of the amino acid to the left of the sulfur in thethioester compounds disclosed in our copending applications, has notbeen determined. It is thought that this amino acid functions as anadditional recognition site for the enzyme. If this is true, it would beexpected that a compound with an amino acid here would be a betterinhibitor. Applicants have found that various amino acids are effectiveand that the hydroxyprolines, proline, L-, and D,L-3,4-dehydroproline,and thiazolidine-4-carboxylic acid derivatives are all effectiveanti-hypertensive agents and have high inhibitory potency for ACE.

SUMMARY OF THE INVENTION

Novel inhibitors of ACE are disclosed which have the general formula

    R--A--S--Z                                                 (I)

wherein,

R is hydrogen, formyl, acetyl, propanoyl, butanoyl, phenylacetyl,phenylpropanoyl, benzoyl, cyclopentanecarbonyl, tert-butyloxycarbonyl,cyclopentanecarbonyl-L-lysyl, pyro-L-glutamyl-L-lysyl, L-arginyl,L-lysyl or pyro-L-glutamyl;

A is phenylalanyl, glycyl, alanyl, tryptophyl, tyrosyl, isoleucyl,leucyl, histidyl, or valyl, the α-amino group thereof being in amidelinkage with R;

S is a sulfur atom in thioester linkage with A and Z; ##STR1## R₁ ishydrogen or halogen; R₁ ' is hydrogen or halogen;

R₂ is hydrogen, lower alkyl or trifluoromethyl;

R₃ is hydrogen, lower alkyl or trifluoromethyl, not more than one of R₂and R₃ being trifluoromethyl, and at least one of R₁, R₁ ', R₂ or R₃ isa halogen or trifluoromethyl substituent;

R₄ is hydrogen, lower alkyl or phenyl-lower alkylene;

R₅ is hydrogen, lower alkyl or phenyl-lower alkylene;

R₆ is hydrogen or hydroxy or when n=2, R₆ can also be halogen;

R₇ is hydrogen, lower alkanoyl or amino(imino)-methyl;

R₈ is hydrogen, lower alkyl or hydroxy-lower alkylene;

R₉ is hydrogen, lower alkyl, phenyl, phenyl-lower alkylene,hydroxy-lower alkylene, hydroxyphenyl-lower alkylene, amino-loweralkylene, guanidino-lower alkylene, mercapto-lower alkylene, loweralkyl-thio-lower alkylene, imidazolyl-lower alkylene, indolyl-loweralkylene, carbamoyl-lower alkylene or carboxy-lower alkylene;

or R₈ and R₉ together form a (CH₂)_(v) bridge which completes a ring of5 or 6 atoms with the nitrogen and carbon to which they are attached,one carbon optionally bearing a hydroxy group when v=4, one carbonoptionally bearing a hydroxy group or halogen group when v=3;

R₁₀ is hydrogen or lower alkyl;

R₁₁ is hydrogen, lower alkyl or lower alkanoyl;

R₁₂ is carboxy, lower alkoxycarbonyl, carbamoyl, N-substituted carbamoylor cyano;

R₁₃ is hydrogen, lower alkyl or phenyl-lower alkylene;

R₁₄ is hydrogen, lower alkyl, phenyl-lower alkylene, hydroxy-loweralkylene, amino-lower alkylene, guanidino-lower alkylene,imidazolyl-lower alkylene, indolyl-lower alkylene, mercapto-loweralkylene, lower alkyl-thio-lower alkylene, carbamoyl-lower alkylene orcarboxy-lower alkylene;

R₁₅ is hydrogen, lower alkyl, phenyl or phenyl-lower alkylene;

R₁₆ is hydrogen, lower alkyl, phenyl or phenyl-lower alkylene;

R₁₇ is hydrogen, hydroxy or lower alkyl or when s=2, R₁₇ can also behalogen;

R₁₈ is hydrogen or lower alkyl;

R₁₉ is lower alkyl;

R₂₀ is lower alkyl;

R₂₁ is hydrogen or lower alkyl;

or R₁₉ and R₂₀ together form a (CH₂)_(w) bridge which completes a ringof 5 atoms with the carbon to which they are attached;

or R₁₉ and R₂₁ together form a (CH₂)_(x) bridge which completes a ringof 5 atoms with the nitrogen and carbon to which they are attached;

R₂₂ is hydrogen or lower alkyl;

R₂₃ is hydrogen or lower alkyl;

R₂₄ is hydroxy, amino or lower alkoxy;

R₂₅ is hydrogen or when m=1, p=0, R₄ =H and R₇ =lower alkanoyl, then R₂₅is hydrogen or lower alkyl;

X is O or S;

m, t and u each is 0 or 1;

n and s each is 1, 2 or 3;

p is 0, 1, 2, 3 or 4;

q and r each is 0, 1 or 2;

v is 3 or 4;

w is 4;

x is 3; and

z is 2 or 3;

with the further provisos that in Formula VIII when R₂₄ is OH, s is 2and R₁₇ is H or OH, (a) if r is 0, R₁₆ may not both be H and R₁₆ may notbe methyl if R₁₅ is H; in formula IX when R₁₉ and R₂₁ togetherconstitute (CH₂)_(x), R₁₈ may not be hydrogen or methyl; in formula X, umust be 1 when R₂₃ is H; and in formula IV, at least one of m and p isother than 0. The asterisks indicate asymmetric carbons.

The disclosed compounds are inhibitors of ACE and are useful asanti-hypertensive agents.

DETAILED DESCRIPTION OF THE INVENTION

The invention in its broad aspects includes thioester compounds whichcontain at least one amino acid and preferably two amino acids. Theseamino acids may be substituted or unsubstituted. The thioester compoundsdisclosed herein all contain one amino acid represented by A in formulaI. It is preferred that the thioester compounds of this inventioncontain an acyl derivative of amino acid A represented by R--A informula I. It is also preferred that a second amino acid is also presentin these thioester compounds. If a second amino acid is present, it isfound in the group represented by Z in formula I.

The amino acid A may be selected from the group comprisingphenylalanine, glycine, alanine, tryptophan, tyrosine, isoleucine,leucine, histidine and valine. Of these amino acids, phenylalanine ispreferred. The preferred acyl derivative R, described above, is in amidelinkage with the α-amino group of the amino acid A. Of these acylderivatives, benzoyl is preferred. Thus, the preferred R--A grouping isNα-benzoyl-phenylalanine. While it is preferred that the amino acid A bein the L-form, it may also be in the D-form or racemic in form.

The remaining portion of the thioester compounds of this invention isrepresented by Z in formula I. Z is selected from the group of compoundshaving formulas II-XI. It is preferred that Z contain an amino acid.Although any amino acid may be utilized, it is preferred that proline,hydroxyproline or 3,4-dehydroproline be utilized. It is preferred thatthe amino acid in Z be in the L-form.

The lower alkyl groups represented by any of the variables includesstraight and branched chain hydrocarbon radicals containing one to sevencarbon atoms. The lower alkylene groups are of the same kind also havingone to seven carbon atoms. Similarly, the lower alkoxy groups are of thesame kind having one to seven carbon atoms with a link to oxygen. Thelower alkanoyl groups are the acyl radicals of the lower fatty acidshaving one to seven carbon atoms. The amino(imino)-methyl grouprepresented by R₇ is the residue of the guanidino radical ##STR2## TheN-substituted carbamoyl of R₁₂ is carbamoyl substituted at the nitrogenposition with a lower alkyl or phenyl-lower alkylene. The halogen may beselected from the group consisting of F, Cl, Br or I.

The compounds of formula I can be produced by various methods ofsynthesis. According to a preferred method, R--A and HS--Z are coupledto produce R--A--S--Z. For this coupling, any conventional couplingagent in preferably an anhydrous medium may be used. In anotherpreferred method, R--A--S is coupled with halo-Z to produce R--A--S--Zusing conventional coupling methods. In a third preferred method,R--A--S is coupled with a vinyl-Z to produce R--A--S--Z by heating. Fora particular desired thioester compound, one method may be morepreferred than the others. Examples of suitable coupling agents are1,1'-carbonyldiimidazole, dicyclohexylcarbodiimide, ethoxyacetylene ordiphenylphosphoryl azide. Examples of suitable anhydrous medium aretetrahydrofuran (THF), dichloromethane, dioxane or dimethylformamide(DMF) although any other suitable anhydrous medium may be used.

The group R--A where R is benzoyl may be obtained commercially or issynthesized using the procedure described in copending application Ser.No. 116,950 incorporated herein by reference. The group R--A where R isformyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl ortert-butyloxycarbonyl (Boc) are commercially available.

Reactants which are commercially available refer to those reactantswhich can be obtained from standard chemical and biochemical supplycompanies. Examples of such companies include Aldrich Chemical Company,Inc., Metucken, N.J. and Sigma Chemical Co., St. Louis, Mo. The groupR--A where R is cyclopentanecarbonyl is synthesized using the proceduredescribed in copending application Ser. No. 064,901 incorporated hereinby reference. The group R--A where R is cyclopentanecarbonyl-L-lysyl orpyro-L-glutamyl-L-lysyl is synthesized using the procedure described incopending application Ser. No. 064,902 incorporated herein by reference.The group R--A where R is L-arginyl, L-lysyl or pyro-L-glutamyl issynthesized using the procedure described in copending application Ser.No. 064,903 incorporated herein by reference.

The HS--Z group can be produced by various methods of synthesis. Forthis description, the synthesis of HS--Z where Z is formula VIII will beutilized for illustration purposes only. The process of forming HS--Zwhere Z is any of the formulas II-XI is done in a similar manner.

According to the preferred method, the imino group of formula VIII, i.e.##STR3## wherein R₁₇, R₂₄ and s are defined above, is acylated with anacid of the formula ##STR4## wherein R₁₅, R₁₆ and r are defined aboveand R₂₆ is hydrogen, lower alkyl, phenyl, substituted phenyl wherein thephenyl substituent is halogen, lower alkyl or lower alkoxy, phenyl-loweralkylene, diphenyl-lower alkylene, triphenyl-lower alkylene, loweralkylthiomethyl, phenyl-lower alkylthiomethyl, lower alkanoylamidomethylor R₂₇ ##STR5## wherein R₂₇ is hydrogen, hydroxy or lower alkyl.Preferably R₂₆ is acetyl. The acylation can be effected in the presenceof a coupling agent in anhydrous medium. Any coupling agent andanhydrous medium may be utilized as previously described. Or the acid offormula XIII can first be activated prior to reaction with the aminogroup of formula XII involving formation of a mixed anhydride,symmetrical anhydride, acid chloride, active ester, Woodward reagent Kor the like. For a review of the methods for acylation see Methoden derOrganischen Chemie (Houben-Weyl), Vol. XV, part II, page 1 et seq.(1974). Deprotection, i.e. removal of R₂₆ when R₂₆ is not H, of theproduct of the acylation of XII with XIII can be effected byconventional means such as treatment with hot trifluoroacetic acid, coldtrifluoromethanesulfonic acid, mercuric acetate, sodium in liquidammonia or the like. For a review of these methods see Methoden derOrganische Chemie (Houben-Weyl), Vol. XV, part I, page 376 et seq.(1974). When R₂₆ is the preferred R₂₇ ##STR6## the product is preferablydeprotected by ammonolysis.

Another method of forming HS--Z as illustrated by Z of formula VIII isto react the amino group of formula XII with ω-haloalkanoic acids of theformula ##STR7## wherein Y is bromo, chloro or iodo to form ##STR8##This product is then subjected to displacement or addition with theanion of a thiol or a thioacid of the formula R₂₆ --SH. The acid offormula XIV is first activated as previously described. The reaction ofXIV and XII is conducted in an alkaline medium, for example alkali metalhydroxide, alkali metal bicarbonate, or alkali metal carbonate. Thereaction of XV with R₂₆ --SH is also conducted in an alkaline medium,preferably alkali metal carbonate. Deprotection is accomplished asdescribed above.

Another method of forming HS--Z as illustrated by Z of formula VIII isto react the amino group of formula XII with thiolactones of the formula##STR9## to yield the desired product HS--Z. This reaction is conductedin an anhydrous medium such as THF or the like.

A variation of this procedure involves the use of an acrylic acid of theformula ##STR10## as starting material. This acrylic acid is firstconverted to the acid halide and reacted with the amino group XII toobtain the following product ##STR11## This product is then subjected tothe addition of a thiol or a thioacid of the formula R₂₆ --SH asdescribed above. The reaction of the acrylic acid with the amino groupof formula XII is conducted in an alkaline medium, preferably an alkalimetal carbonate.

Alternatively, the acrylic acid of formula XVII can be reacted with athioacid of the formula R₂₆ --SH to form ##STR12## which is converted tothe acid halide and reacted with the amino group of formula XII.

When an acid of the imino group of formula XII, i.e. when R₂₄ ishydroxy, is used as starting material, the final product obtained as thefree carboxylic acid can then be converted to its ester, for example byesterification with a diazoalkane, like diazomethane,1-alkyl-3-p-tolyltriazene, like 1-n-butyl-3-p-tolyltriazene or the like.Treatment of the ester, preferably the methyl ester, with an alcoholicammonia solution, converts the free acid to the amide, i.e. R₂₄ is NH₂.When an ester of the amino group of formula XII is used as startingmaterial, the final product obtained can be treated with trifluoroaceticacid and anisole to remove the ester group (R₂₄) to yield the freecarboxylic acid.

The thioester compounds of the formula A--S--Z, i.e. R is hydrogen, isprepared preferably by deprotecting the thioester compounds N.sup.α-tert-butyloxycarbonyl-A--S--Z. One method of deprotecting thesecompounds is described in copending application Ser. No. 064,899incorporated herein by reference.

Where Z is defined by formula XI, the HS--Z compounds are preferablysynthesized by reacting the amino group with a haloalkylsulfonyl halidein an organic base such as N,N-dimethylaniline, N-methylmorpholine orthe like in an inert organic solvent such as THF, dioxane,dichlormethane or the like. The product from this reaction is reactedwith R₂₆ --SH in the presence of an organic base and organic solvent asdescribed above to yield HS--Z (XI).

The amino group of Z defined by formula III is derived fromnitrosoazetidine-2-carboxylic acid, nitrosoprolines or nitrosopipecolicacids which have the formula ##STR13## and which are prepared from thecorresponding azetidine-2-carboxylic acid, proline or pipecolic acid,respectively, by means of nitroxyl tetrafluoroborate according to themethod of Lijinsky et al., Tetrahedron 26, 5137 (1970). They can also beproduced by the method described by Nagasawa et al., J. Med. Chem. 16,583 (1973).

The nitroso amino acid of formula III is next reduced to the N-aminoderivative which has the formula ##STR14## e.g., with zinc-acetic acidby the method described by Klosterman et al., Biochemistry 6, 173(1967).

The R--A--S group can be synthesized by various methods. For thisdescription, the synthesis of R--A--S where R is benzoyl and A isphenylalanyl will be utilized for illustration purposes only.

According to the preferred method thiophenol is coupled to N.sup.α-tert-butyloxycarbonylphenylalanine using conventional coupling agentsto give ##STR15## It is preferred that this be done by the mixedanhydride method in ethyl acetate. This compound is deprotected byreacting the compound in TFA and anisole followed by hydrogen chloridein ethanol to produce the hydrogen chloride salt of ##STR16## Thislatter compound is preferably reacted with benzoyl chloride in sodiumcarbonate and ethyl acetate to produce ##STR17## The thiophenol group isremoved by reacting with NaSH in ethanol under nitrogen to produceN.sup.α -benzoyl-Phe-SH.

Another method of producting N.sup.α -benzoyl-Phe-SH is to react N.sup.α-benzoyl-Phe with H₂ S in a mixed anhydride reaction to produce thedesired product. A third method includes the reaction of N.sup.α-Boc-Phe with H₂ S in a mixed anhydride reaction as described above.This is then followed by a reaction with hydrogen chloride in ethanoland by a reaction with benzoyl chloride as described above.

The N.sup.α -benzoyl-Phe-SH is then reacted with the compound of FormulaXVIII, for example, by heating in preferably toluene to produce N.sup.α-benzoyl-Phe-S-Z. In addition, the N.sup.α -benzoyl-Phe-SH can bereacted with the compound of Formula XV to produce N.sup.α-benzoyl-Phe-S-Z. For a more detailed description of the above-describedmethods, see copending application Ser. No. 128,953 filed on Mar. 10,1980 now aband.

The thioester compounds of formula I have one or more asymmetriccarbons. The following compound using R=benzoyl, A=Phe and Z of formulaVIII is used for illustration purposes only. In the compound, ##STR18##the possible asymmetric carbons are indicated by an asterisk. When R₁₅,R₁₆ or R₁₇ is other than hydrogen the carbon to which it is attached isasymmetric. The other carbons marked by an asterisk above areasymmetric. The compounds accordingly exist in sterioisomeric forms orin racemic mixtures thereof. All of these are within the scope of theinvention. The above described syntheses can utilize the racemate or oneof the enantiomers as starting material. When the racemic startingmaterial is used in the synthetic procedure or a racemic mixture resultsfrom the synthesis, the stereoisomers obtained in the product can beseparated by conventional chromatographic or fractional crystallizationmethods. In general, the L-isomer with respect to the carbon of theamino group constitutes the preferred isomeric form. Also the D-isomerwith respect to the α-carbon in the acyl side chain (e.g. the carbonbearing R₁₆ in the above example) is preferred.

The compounds of this invention form basic salts with various inorganicand organic bases which are also within the scope of the invention. Suchsalts include ammonium salts, alkali metal salts like sodium andpotassium salts (which are preferred), alkaline earth metal salts likethe calcium and magnesium salts, salts with organic bases, e.g.,dicyclohexylamine salt, benzathine, N-methyl-D-glucamine, hydrabaminesalts, salts with amino acids like arginine, lysine and the like. Thenon-toxic, physiologically acceptable salts are preferred, althoughother salts are also useful, e.g., in isolating or purifying theproduct, as illustrated in the examples in the case of thedicyclohexylamine salt.

The salts are formed in conventional manner by reacting the free acidform of the product with one or more equivalents of the appropriate baseproviding the desired cation in a solvent or medium in which the salt isinsoluble, or in water and removing the water by freeze drying. Byneutralizing the salt with an insoluble acid like a cation exchangeresin in the hydrogen form (e.g., polystyrene sulfonic acid resin likeDowex 50) or with an aqueous acid and extraction with an organicsolvent, e.g., ethyl acetate, dichloromethane or the like, the free acidform can be obtained, and, if desired, another salt formed.

Additional experimental details are found in the examples which arepreferred embodiments and also serve as models for the preparation ofother members of the group.

The compounds of this invention inhibit the conversion of thedecapeptide angiotensin I to angiotensin II and therefore are useful inreducing or relieving angiotensin related hypertension. The action ofthe enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma,produces angiotensin I. Angiotensin I is converted by angiotensinconverting enzyme (ACE) to angiotensin II. The latter is an activepressor substance which has been implicated as the causative agent invarious forms of hypertension in various mammalian species, e.g., ratsand dogs. The compounds of this invention intervene in the ##STR19##sequence by inhibiting angiotensin converting enzyme and reducing oreliminating the formation of the pressor substance angiotensin II. Thusby the administration of a composition containing one or a combinationof compounds of formula I or physiologically acceptable salt thereof,angiotensin dependent hypertension in the species of mammal sufferingtherefrom is alleviated. A single dose, or preferably two to fourdivided daily doses, provided on a basis of about 0.1 to 100 mg. perkilogram per day, preferably about 1 to 50 mg. per kilogram per day isappropriate to reduce blood pressure as indicated in the animal modelexperiments described by S. L. Engel, T. R. Schaeffer, M. H. Waugh andB. Rubin, Proc. Soc. Exp. Biol. Med. 143, 483 (1973). The substance ispreferably administered orally, but parenteral routes such assubcutaneous, intramuscular, intravenous or intraperitoneal can also beemployed.

The compounds of this invention can be utilized to achieve the reductionof blood pressure by formulating in compositions such as tablets,capsules or elixirs for oral administration or in sterile solutions orsuspensions for parenteral administration. About 10 to 500 mg. of acompound or mixture of compounds of formula I or physiologicallyacceptable salt is compounded with a physiologically acceptable vehicle,carrier, excipient, binder, preservative, stabilizer, flavor, etc., in aunit dosage form as called for by accepted pharmaceutical practice. Theamount of active substance in these compositions or preparations is suchthat a suitable dosage in the range indicated is obtained.

Illustrative of the adjuvants which may be incorporated in tablets,capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alignic acid and the like; a lubricant such as magnesiumstearate; a sweetening agent such as sucrose, lactose or saccharin; aflavoring agent such as peppermint, oil of wintergreen or cherry. Whenthe dosage unit form is a capsule, it may contain in addition tomaterials of the above type a liquid carrier such as a fatty oil.Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice by dissolving or suspending theactive substance in a vehicle such as water for injection, a naturallyoccurring vegetable oil like sesame oil, coconut oil, peanut oil,cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate orthe like. Buffers, preservatives, and the like can be incorporated asrequired.

The present invention will be further described by the followingexamples. All temperatures are in degrees Celsius unless otherwiseindicated.

Examples 1-15 describe the synthesis of R--A.

EXAMPLE 1 Preparation of N.sup.α -Benzoylphenylalanine

A mixture containing 8.21 of phenylalanine, 5.565 g of Na₂ CO₃ in 40 mlof water and 20 ml of tetrahydrofuran (THF) was stirred at roomtemperature. Benzoyl chloride, 7.73 g, dissolved in 20 ml of anhydrousTHF, was added gradually over a period of 45 minutes with continuedstirring at room temperature. Stirring was allowed to continue for anadditional hour, at which time the reaction mixture was transferred to arotary evaporator at 30° C. to remove the THF. An excess of water wasthen added and the reaction mixture extracted four times with ethylacetate. The aqueous phase was then titrated to pH 2 with 3N HCl. Awhite crystalline precipitate formed which was recovered by filtration,washed three times with cold dilute HCl and three times with cold water,and dried in a vacuum oven over P₂ O₅ at about 50° C. The product wasrecrystallized from aqueous ethanol, yielding 8.37 g, m.p. 183° C.-184°C., which migrated as a single compound on thin layer chromatography infive separate solvent systems. In this reaction sequence the racemate isobtained.

If a NaOH solution is used in place of the Na₂ CO₃ in Example 1 andsubstantially following the procedure set forth in Carter, H. E. et al.,J. Biol. Chem. 138, 627 (1941), optical activity is maintained. That isif L-Phe or D-Phe is the starting material, N.sup.α -benzoyl-L-Phe orN.sup.α -benzoyl-D-Phe respectively is produced.

Similarly, if benzoyl N-hydroxysuccinimide ester is used in place ofbenzoyl chloride in Example 1 and substantially following the procedureof Example 1, the optical activity is maintained.

EXAMPLE 2

By substituting glycine, alanine, tryptophan, tyrosine, isoleucine,leucine, histidine or valine for Phe in Example 1 and substantiallyfollowing the procedure of Example 1, the N.sup.α -benzoyl derivativesof these amino acids are obtained.

EXAMPLE 3

Although the formyl, acetyl, propanoyl, butanoyl, phenylacetyl,phenylpropanoyl or tert-butyloxycarbonyl derivatives of the Phe, Gly,Ala, Trp, Tyr, Ile, Leu, His or Val are commercially available, they arealso obtained by substituting the appropriate acyl chloride or acylN-hydroxy succinimide ester for the benzoyl chloride in Examples 1 and 2and substantially following the procedure of Example 1.

EXAMPLE 4 Synthesis of N.sup.α -cyclopentanecarbonylphenylalanine

A cool solution of 2.06 gm of dicyclohexylcarbodiimide in 10 ml ofdichloromethane was added to a solution of 1.4114 gm ofcyclopentanecarboxylic acid in 5 ml of dichloromethane at -5° C. It wasfollowed by the addition of 4.28 gm of phenylalanine benzyl estertouluenesulfonate salt in 10 ml of DMF which was neutralized with 1.36ml of N-ethyl morpholine. The reaction mixture was stirred at 0° C. forone hour and then at room temperature for three hours. Dicyclohexylureawas removed by filtration and 50 ml of ethyl acetate was added to thefiltrate. The organic phase was washed until neutral, dried overanhydrous MgSO₄ and filtered. The solvent was removed with a rotaryevaporator. The residue was crystallized from isopropanol and hexaneyielding 2.35 gm of white crystals having a melting point of 88°-89° C.Elemental analysis of these crystals yielded the following.

Calculated: C=75.19; H=7.17; N=3.9855. Found: C=74.96; H=7.17; N=4.09.

The benzyl ester was removed by hydrogenolysis with 2 gm of 10%palladium on carbon in absolute alcohol. The catalyst was removed byfiltration and the ethanol was removed by a rotary evaporator. Theresidue was crystallized from ether and hexane yielding 1.15 gm whitecrystals of the named product having a melting point of 107°-108° C.Elemental analysis of these crystals yielded the following.

Calculated: C=68.94; H=7.33; N=5.36. Found: C=68.90; H=7.32; N=5.34.

The product was found to be homogeneous by paper electrophoresis and byTLC in three separate solvent systems. The named product may beabbreviated as N.sup.α -cpc-L-Phe.

EXAMPLE 5

By substituting Gly, Ala, Trp, Tyr, Ile, Leu, His or Val for the Phe ofExample 4 and substantially following the procedure of Example 4, theN.sup.α -cpc derivatives of these amino acids are obtained.

EXAMPLE 6 Synthesis of the N-hydroxysuccinimide ester of cyclopentanecarboxylic acid

A cool solution of 11.4 gm of dicyclohexylcarbodiimide indimethylformamide (DMF) was added drop-wise to a mixture of 5.71 gm ofcyclopentanecarboxylic acid and 5.76 gm of N-hydroxysuccinimide in DMFat 0° C. The reaction mixture was stirred at 0° C. for 30 minutes andthen at 4° C. overnight. Crystalline dicyclohexylurea was removed byfiltration and the precipitate was washed with ethyl acetate. Solventsfrom the combined filtrate were removed under reduced pressure and theresidue was crystallized from benzene and hexane yielding 5.77 gm ofwhite crystals having a melting point of 72.5°-73° C. The infraredabsorption spectrum in chloroform gave a typical spectrum ofN-hydroxysuccinimide esters. Elemental analysis of the crystals yieldedthe following results.

Calculated: C=56.86; H=6.20; N-6.63. Found: C=56.77; H=6.07; N=6.66.

EXAMPLE 7 Synthesis of N.sup.α -cyclopentanecarbonyl-N.sup.ε-tert-butyl-oxycarbonyl-L-lysine

A solution of 1.2316 gm of N.sup.ε -tert-butyloxycarbonyl-L-lysine and420 mg of NaHCO₃ in 10 ml of water and 5 ml of THF was cooled in an icebath with stirring. To this solution was added a cold solution of 1.19gm of the product from Example 6 in 10 ml of THF. The THF was removedwith a rotary evaporator at 35° C. after the reaction mixture had beenstirred overnight at room temperature. About 20 ml of water was added tothe reaction mixture and the pH was adjusted to 9 with solid NaHCO₃. Theaqueous phase was extracted three times with ethyl acetate and theorganic phase was discarded. The aqueous solution was cooled in an icebath and then acidified to pH 2 with 1N HCl in the presence of ethylacetate. The organic phase was washed twice with ice water and thentwice with a solution of saturated NaCl. The organic solution was driedover anhydrous MgSO₄ and then filtered. The solvent was removed with arotary evaporator and the residue was crystallized from ether and hexaneyielding 1.135 gm of white crystals having a melting point of104.5°-105.5° C. Elemental analysis of the crystals yielded thefollowing.

Calculated: C=59.63; H=8.83; N-8.18. Found: C=59.74; H=8.85; N=8.24.

The product was shown to be homogeneous by paper electrophoresis and byTLC with three separate solvent systems.

EXAMPLE 8 Synthesis of N.sup.α -cyclopentanecarbonyl-N.sup.ε-tert-butyloxycarbonyl-L-lysine-N-hydroxysuccinimide ester

A solution of 1.027 gm of the product from Example 7 and 346 mg ofN-hydroxysuccinimide in 10 ml of CH₂ Cl₂ was cooled to -5° C. To thissolution was added with stirring a cold solution of 680 mg ofdicyclohexylcarbodiimide in 5 ml of CH₂ Cl₂. The reaction mixture wasstirred at 0° C. for 30 minutes and then at 4° C. overnight. Crystallinedicyclohexylurea was removed by filtration and was washed with ethylacetate. The combined filtrate was washed twice with a 1.0N NaHCO₃,twice with water and finally with a solution of saturated NaCl. Theorganic phase was dried over anhydrous MgSO₄, filtered, and the solventwas removed with a rotary evaporator. The residue was crystallized fromisopropanol yielding 0.844 gm of white crystals having a melting pointof 140.5° C. The infrared absorption spectrum in chloroform gave atypical spectrum of N-hydroxysuccinimide esters. Elemental analysis ofthe crystals yielded the following.

Calculated: C=57.39; H=7.57; N=9.56. Found: C=57.10; H=7.57; N=9.61.

EXAMPLE 9 Synthesis of N.sup.α -cyclopentanecarbonyl-N.sup.ε-tert-butyloxycarbonyl-L-lysylphenylalanine

A solution of 220 mg of the product from Example 8 in 2 ml of dioxanewas added drop-wise to a mixture of 99.1 mg of phenylalanine and 51 mgof NaHCO₃ in a mixture of 2 ml of water and 1 ml of DMF. The reactionmixture was stirred at room temperature overnight and dioxane wasremoved with a rotary evaporator at 35° C. Ethyl acetate (10 ml) wasadded to the reaction mixture, cooled, acidified to pH 2 with 0.1N HCland the aqueous solution was discarded. The organic phase was washedwith ice water, a solution of saturated NaCL, and dried over anhydrousMgSO₄ ; and the solvent was removed with a rotary evaporator. Theresidue was crystallized from ether and petroleum ether (b.p. 30°-60°C.) yielding 95 mg of white solid having a melting point of 90°-92° C.The product was shown to be homogeneous by paper electrophoresis and byTLC in four separate solvent systems. Elemental analysis yielded thefollowing results.

Calculated: C=63.78; H=8.03; N=8.58. Found: C=63.40; H=8.07; N=8.34.

The named compound can be abbreviated N.sup.α -cpc-N.sup.ε-Boc-L-Lys-L-Phe.

EXAMPLE 10

By substituting Gly, Ala, Try, Tyr, Ile, Leu, His or Val for the Phe inExample 9 and substantially following the procedure of Example 9, theN.sup.α -cpc-N.sup.ε -Boc-L-Lys derivatives of these amino acids areobtained.

EXAMPLE 11

By substituting pyro-L-glutamic acid for the cyclopentanecarboxylic acidin Example 6 and substantially following the procedures of Examples 6-8,N.sup.α -pyro-L-glutamyl-N.sup.ε-tert-butyloxycarbonyl-L-lysine-N-hydroxysuccinimide ester is obtained.By substituting this product for the N.sup.α -cpc-N.sup.ε-Boc-L-Lys-N-hydroxysuccinimide ester in Examples 9 and 10 andsubstantially following the procedures of Example 9, the N.sup.α-pyro-L-glutamyl-N.sup.ε -Boc-L-Lys derivatives of the amino acids areobtained.

EXAMPLE 12 Preparation of pyro-L-glutamylphenylalanine benzyl ester

A solution of 0.52 g of pyro-L-glutamic acid, 1.72 g of phenylalaninebenzyl ester toluene sulfonic acid and 0.55 mg of N-ethylmorpholine in 5ml of dimethylformamide (DMF) and 20 ml of dichloromethane was cooled inan ice bath with stirring. A solution of 0.826 g ofdicyclohexylcarbodiimide in 2 ml dichloromethane was added to the abovereaction mixture. The reaction mixture was stirred in an ice water bathfor 1 hour and then at room temperature overnight. Dicyclohexylurea wasremoved by filtration and the product was washed in ethyl acetate.Solvents of the combined filtrates were removed under reduced pressurewith a rotary evaporator at 40°. Ethyl acetate (25 ml) was added to theresidue and the organic solution was washed unit neutral. The organicphase was dried over anhydrous MgSO₄, filtered and then the solvent wasremoved with a rotary evaporator. The material was crystallized fromisopropanol and ether, yield; 1.01 g of white needles, m.p. 103°-104.5°C. The material was shown to be homogeneous by paper electrophoresis andby TLC with five separate solvent systems. Elemental analysis yieldedthe following results:

Calculated: C=68.84; H=6.05; N=7.64. Found: C=68.58; H=6.05; N=7.56.

EXAMPLE 13 Preparation of pyro-L-glutamylphenylalanine

The benzyl ester protecting group of the compound of Example 12 (1.0 g)was removed by catalytic hydrogeneolysis with 150 mg of 10% (by weight)Pd on carbon in 0.15 ml of glacial acetic acid and 15 ml of ethanol at20 psi of H₂ at room temperature overnight. The catalyst was removed byfiltration. Solvent was removed with a rotary evaporator. The materialwas crystallized from isopropanol and benzene, to yield a total of 402mg of white crystals, m.p. 147°-149° C. The material was shown to behomogeneous by paper electrophoresis and TLC with three separate solventsystems. Elemental analysis yielded the following results.

Calculated: C=60.86; H=5.84; N=10.14. Found: C=60.37; H=5.85; N=9.98.

EXAMPLE 14

By substituting N.sup.α, N.sup.ε -bis-t-butyloxycarbonyl-L-lysine(hereinafter bis-Boc-L-Lys) or N⁶⁰ , N.sup.α, N.sup.ω-triadamantyloxycarbonyl-L-arginine (hereinafter tri-Adoc-L-arginine)for pyro-L-glutamic acid in Example 12 and by following substantiallythe procedure of Examples 12 and 13, the corresponding bis-Boc-L-Lys ortri-Adoc-L-Arg derivatives of Phe will be synthesized. Bis-Boc-L-Lys iscommercially available. Tri-Adoc-L-Arg is prepared according to Jager,G. and Geiger, R., Chem. Ber. 102, 1727 (1970).

EXAMPLE 15

By substituting the benzyl esters of Gly, Ala, Trp, Tyr, Ile, Leu, Hisor Val, for Phe in Examples 12 and 14 and substantially following theprocedures of Examples 12, 13 and 14, the corresponding pyro-L-glutamyl,bis-Boc-L-Lys and tri-Adoc-L-Arg derivatives of these amino acids areobtained.

Examples 16-26 described the synthesis of HS--Z where Z is defined byformula VIII. The procedures followed in these examples are described inU.S. Pat. Nos. 4,046,889 and 4,105,776.

EXAMPLE 16 Synthesis of N-(2-Benzoylthioacetyl)-L-Proline

L-Proline (5.75 g.) is dissolved in 1.0N sodium hydroxide (50 ml.) andthe solution is chilled in an ice-water bath. Sodium hydroxide 2N (26ml.) and chloroacetyl chloride (5.65 g.) are added and the mixture isstirred vigorously at room temperature for 3 hours. A suspension ofthiobenzoic acid (7.5 g.) and potassium carbonate (4.8 g.) in water (50ml.) is added. After 18 hours stirring at room temperature, the reactionmixture is acidified and extracted with ethyl acetate. The ethyl acetatelayer is washed with water, dried over magnesium sulfate andconcentrated to dryness in vacuo. The residue (14.6 g.) is dissolved inethyl acetate (150 ml.) and dicyclohexylamine (11 ml.) is added. Thecrystals are filtered and recrystallized from ethyl acetate, yield 5.7g. m.p. 151°-152°. To convert the salt to the acid, the crystals aredissolved in a mixture of 5% aqueous potassium bisulfate (100 ml.) andethyl acetate (300 ml.). The organic phase is washed once with water,dried over magnesium sulfate and concentrated to dryness in vacuo, yield3.45 g.

EXAMPLE 17 Synthesis of N-(2-Mercaptoacetyl)-L-Proline

N-(2-Benzoylthioacetyl)-L-proline (3.4 g.) is dissolved in a mixture ofwater (10.5 ml.) and concentrated ammonia (6.4 ml.). After 1 hour, thereaction mixture is diluted with water and filtered. The filtrate isextracted with ethyl acetate and then acidified with concentratedhydrochloric acid, saturated with sodium chloride and extracted twicewith ethyl acetate. The ethyl acetate extracts are washed with saturatedsodium chloride and concentrated to dryness, yield 1.5 g. The product,N-(2-mercaptoacetyl)-L-proline is crystallized from ethyl acetate (m.p.133°-135°).

EXAMPLE 18 Synthesis of N-(2-Benzoylthioacetyl)-L-Proline Methyl Ester

N-(2-Benzoylthioacetyl)-L-proline obtained in Example 16, is dissolvedin methanol and an ethereal solution of diazomethane is added untilthere is a persistent yellow color. After 15 minutes, a few drops ofacetic acid are added and the solvent is removed in vacuo to obtainN-(2-benzoylthioacetyl)-L-proline methyl ester.

EXAMPLE 19 Synthesis of N-(2-Mercaptoacetyl)-L-Proline Amide

The product of Example 18 is dissolved in 10% methanolic ammonia and thesolution is stored at room temperature in a pressure bottle. When thinlayer chromatographic analysis indicates that the two ester functionshave been ammonolyzed, the reaction mixture is concentrated to drynessto obtain N-(2-mercaptoacetyl)-L-proline amide.

EXAMPLE 20 Synthesis of N-(3-Mercaptopropanoyl)-L-proline tert-butylEster

To a solution of L-proline tert-butyl ester (3.42 g.) in drytetrahydrofuran (10 ml.) chilled in an ice bath, propiothiolacetone(1.76 g.) is added. After 5 minutes storage in the ice bath and 3 hoursat room temperature, the reaction mixture is diluted with ethyl acetate(200 ml.) and washed with 5% potassium bisulfate, and water. The organiclayer is dried over magnesium sulfate and concentrated to dryness invacuo. The residue N-(3-mercaptopropanoyl)-L-proline tert-butyl ester iscrystallized from ether-hexane, yields 3.7 g., m.p. 57°-58°.

EXAMPLE 21 Synthesis of 3-Acetylthio-2-Methylpropanoic Acid

A mixture of thioacetic acid (50 g.) and methacrylic acid (40.7 g.) isheated on the steam bath for 1 hour and then stored at room temperaturefor 18 hours. After confirming by nmr spectroscopy that completereaction of the methacrylic acid has been achieved, the reaction mixtureis distilled in vacuo and the desired 3-acetylthio-2-methylpropanoicacid is separated in the fraction with boiling point 128.5°-131° (2.6mmHg.), yield 64 g.

EXAMPLE 22 Synthesis of N-(3-Mercapto-2-methylpropanoyl)-L-Prolinetert-butyl Ester

L-Proline tert-butyl ester (5.1 g.) is dissolved in dichloromethane (40ml.) and the solution stirred and chilled in an ice bath.Dicyclohexylcarbodiimide (6.2 g.) dissolved in dichloromethane (15 ml.)is added followed immediately by a solution of3-acetylthio-2-methylpropanoic acid (4.9 g.) in dichloromethane (5 ml.).After 15 minutes stirring in the ice bath and 16 hours at roomtemperature, the precipitate is filtered off and the filtrate isconcentrated to dryness in vauco. The residue is dissolved in ethylacetate and washed neutral. The organic phase is dried over magnesiumsulfate and concentrated to dryness in vacuo. The residue,N-(3-acetylthio-2-methylpropanoyl)-L-proline tert-butyl ester, ispurified by column chromatography (silica gel, chloroform), yield 7.9 g.The named product is obtained by following the procedure of Example 17.

EXAMPLE 23 Synthesis of N-(3-mercapto-2-D,L-methylpropanoyl)-L-proline

Methacryloyl chloride (4.16 g.) is added to a solution of L-proline(3.45 g.) in a mixture of water (100 ml.) and sodium bicarbonate (12 g.)chilled in an ice water bath, with vigorous stirring. When the additionis completed, the mixture is stirred at room temperature for two hours,and then extracted with ether. The aqueous phase is acidified with 1.0Nhydrochloric acid and extracted with ethyl acetate. The organic phase isconcentrated to dryness in vacuo, the residue is mixed with thiolaceticacid (3.5 g.), a few crystals of azobisisobutyronitrile are added andthe mixture is heated on the steam bath for two hours. The reactionmixture is dissolved in benzene acetic acid (75:25), and applied to acolumn of silica gel. Elution with the same solvent mixture yields theN-(3-acetylthio-2-D,L-methylpropanoyl)-L-proline. The named product isobtained by following the procedure of Example 17.

EXAMPLE 24 Synthesis ofN-[3-(Acetylthio)-2-methylpropanoyl]-D,L-Pipecolic Acid

6.5 g. (0.05 mole) of pipecolic acid are suspended in dimethylacetamide(200 mg.), 9.0 g. (0.05 mole) of 3-acetylthio-2-methylpropanoyl chlorideis added dropwise. The temperature rises to 29° and a clear solutionforms. Then 10.1 g. of N-methylmorpholine is added all at once and thetemperature rises to 34°. The mixture is heated on a steam bath for 1hour when a clear solution forms. This is allowed to stand at roomtemperature overnight and the solid which precipitates is filtered toyield 6.1., m.p. 203°-204°. The solvent is removed and the viscousresidue is triturated with water and 20% HCl. The yellow oil isextracted with 3×150 ml. of ethyl acetate. The ethyl acetate extractsare dried over magnesium sulfate and removed to yield 14 g. ofN-[3-(acetylthio)-2-methylpropanoyl]-D,L-pipecolic acid as a viscousoil.

EXAMPLE 25 Synthesis of N-[3-Mercapto-2-methylpropanoyl]-D,L-PipecolicAcid

Aqueous NH₄ OH (30 ml. water and 20 ml. conc. NH₄ OH) is stirred undernitrogen at 10° for 145 minutes. This is added to 13.0 g. (0.05 m) ofN-[3-(acetylthio)-2-methylporpanoyl]-D,L -pipecolic acid and theresulting solution is stirred for 10 minutes under nitrogen; then atroom temperature for 50 minutes. It is then treated with water and 20%NaCl and the yellow oil extracted with 3×150 ml. of ethyl acetate. Theethyl acetate extract is dried over magnesium sulfate and removed toyield 11.1 g. N-(3-mercapto-2-methylpropanoyl)-D,L-pipecolic acid as aviscous oil. R_(f) 0.62 [silica gel, benzene, acetic acid (7:2)].

EXAMPLE 26

By substituting the appropriate activated acyl group for thechloroacetyl chloride of Example 16 and by substituting the appropriateamino group for L--Pro of Example 16 and substantially following theprocedures of Examples 16-19, the mercapto compounds, HS--Z (VIII),defined in Table 1 are obtained.

                  TABLE 1                                                         ______________________________________                                        R.sub.15    R.sub.16 R.sub.17  R.sub.24                                                                             r   s                                   ______________________________________                                         (1) H          H        H, 3-OH OH     1   2                                  (2) H          H        H       OH     1   1                                  (3) H          H        H       OH     1   3                                  (4) H          H        OH      OH     1   1                                  (5) H          C.sub.2 H.sub.5                                                                        H       OH     1   2                                  (6) CH.sub.3   H        Cl      OC.sub.2 H.sub.5                                                                     1   2                                  (7) CH.sub.3   CH.sub.3 H       OH     1   2                                  (8) CH.sub.3   C.sub.6 H.sub.5 CH.sub.2                                                               H       OH     1   2                                  (9) C.sub.2 H.sub.5                                                                          H        H       OH     1   1                                 (10) H          C.sub.4 H.sub.9                                                                        H       OH     1   3                                 (11) H          H        H       NH.sub.2                                                                             1   2                                 (12) H          H        H, 3-F  OH     2   2                                 (13) H, CH.sub.3                                                                              H        H, 3-OH OH     2   2                                 (14) CH.sub.3, CH.sub.3                                                                       H        H       OH     2   1                                 (15) H          C.sub.2 H.sub.5                                                                        H       OH     2   3                                 (16) H, C.sub.2 H.sub.5                                                                       CH.sub.3 H       OCH.sub.3                                                                            2   2                                 (17) H          CH.sub.3 H, H, 3-OH                                                                            OCH.sub.3                                                                            2   3                                 (18) --         H        H       OH     0   1                                 (19) --         H        H, H, 4-OH                                                                            OH     0   3                                 (20) --         CH.sub.3 H       OC.sub.2 H.sub.5                                                                     0   1                                 (21) --         CH.sub.3 H       NH.sub.2                                                                             0   3                                 (22) --         C.sub.4 H.sub.9                                                                        H, 4-Br OH     0   2                                 (23) --         H        H, 4-CH.sub.3                                                                         OH     0   2                                 (24) --         CH.sub.3 OH      OH     0   1                                 (25) C.sub.3 H.sub.7                                                                          H        H       NH.sub.2                                                                             1   2                                 (26) H          CH.sub.3 H, H, 5-OH                                                                            OH     1   3                                 (27) H          CH.sub.3 H       OH     2   2                                 (28) C.sub.6 H.sub.5 --C.sub.2 H.sub.5                                                        H        H, 4-OH OC.sub.3 H.sub.7                                                                     1   2                                 (29) CH.sub.3   H        C.sub.2 H.sub.5                                                                       NH.sub.2                                                                             1   1                                 (30) H, C.sub.5 H.sub.11                                                                      CH.sub.3 H, 3-C.sub.4 H.sub.9                                                                  OH     2   2                                 ______________________________________                                    

Examples 27-29 described the synthesis of SH--Z where Z is defined byformula XI. The procedures followed in these examples are described inU.S. Pat. No. 4,070,361.

EXAMPLE 27 Synthesis of N-[[2-(Acetylthio)ethyl]sulfonyl]-L-proline a.N-(Vinylsulfonyl)-L-proline t-butyl ester

L-Proline t-butyl ester (6.9 g. 0.04 mol.) and triethylamine (14 ml.,0.1 mol.) are dissolved in 200 ml. of dichloromethane and stirred in anice bath while 2-chloroethanesulfonyl chloride (8.2 g., 0.05 mol.) in100 ml. of dichloromethane is added over 20 minutes. After stirring 2hours, the mixture is washed with 5% potassium bisulfate solution,saturated sodium bicarbonate solution and brine, then evaporated invacuo. The semi-solid residue in chromatographed on 350 ml. silica gelusing 1:1 ethyl acetate/hexane as eluant. The main fraction, comprisingN-(vinylsulfonyl)-L-proline t-butyl ester is crystallized fromether/hexane, m.p. 84°-87° (7.1 g., 68%).

b. N-[[2-(Acetylthio)ethyl]sulfonyl]-L-proline t-butyl ester

N-(Vinylsulfonyl)-L-proline t-butyl ester (5.0 g., 0.0192 mol.),triethylamine (2.8 ml., 0.02 mol.) and thiolacetic acid (1.43 ml., 0.02mol.) are mixed in 100 ml. of ether and allowed to stand overnight. Themixture is washed with 5% potassium bisulfate solution, saturated sodiumbicarbonate solution and brine, then evaporated in vacuo to a yellowoil. the procedure is repeated using half of the above quantities oftriethylamine and thiolacetic acid. Workup as in part (a) affords thecrude product, N-[[2-(acetylthio)ethyl]sulfonyl]-L-proline t-butylester, which is filtered through a short silica gel column andcrystallized from ether/hexane, m.p. 46°-50° (2.9 g., 45%).

c. N-[[2-Acetylthio)ethyl]sulfonyl]-L-proline

The t-butyl ester from part (b) (2.9 g., 0.0086 mol.) is dissolved in 15ml. of anisole and 45 ml. of trifluoroacetic acid and let stand 1 hour.The mixture is evaporated in vacuo to a gummy residue which is taken upin ethyl acetate and treated with a large volume of hexane. Thesupernatant is decanted, and the procedure repeated. The resultingsemisolid is crystallized from ethyl acetate-hexane, m.p. 63°-67°.

EXAMPLE 28 Synthesis of N-[(2-Mercaptoethyl)sulfonyl]-L-proline

N-[[2-(Acetylthio)ethyl]sulfonyl]-L-proline (640 mg., 0.0023 mol.) isdissolved in 5 ml. of water and 5 ml. of concentrated ammonia andstirred 1 hour under nitrogen. The solution is acidified withconcentrated hydrochloric acid, extracted with ethyl acetate, and theextracts are washed with brine, dried (MgSO₄) and evaporated to an oilyresidue which is applied to a 75 ml. silica gel column. Elution with 10%acetic acid/benzene affords a main fraction which is crystallized fromchloroform/hexane, to obtain 440 mg. (81%) of1-[(2-mercaptoethyl)sulfonyl]-L-proline, m.p. 99°-101°.

EXAMPLE 29

By substituting the appropriate haloalkylsulfonyl halide for the2-chloroethanesulfonyl chloride of Example 27 and by substituting theappropriate amino group for the L-Pro-t-butyl ester of Example 27 andsubstantially following the procedures of Examples 27-28, the mercaptocompounds, HS--Z (XI), defined in Table 2 are obtained.

                  TABLE 2                                                         ______________________________________                                                   R.sub.10 R.sub.23    z                                             ______________________________________                                        (1)          CH.sub.3   H           2                                         (2)          H          CH.sub.3    2                                         (3)          C.sub.3 H.sub.7                                                                          H           2                                         (4)          C.sub.2 H.sub.5                                                                          C.sub.2 H.sub.5                                                                           2                                         (5)          C.sub.4 H.sub.9                                                                          H           2                                         (6)          H          H           3                                         (7)          CH.sub.3   C.sub.5 H.sub.11                                                                          3                                         (8)          CH.sub.3   H           3                                         (9)          C.sub.4 H.sub.9                                                                          CH.sub.3    3                                         (10)         CH.sub.3   CH.sub.3    3                                         ______________________________________                                    

Examples 30--35 describe the synthesis of HS--Z where Z is defined byformula II. The procedures followed in these examples are described inU.S. Pat. No. 4,154,935.

EXAMPLE 30 Synthesis of 3-Acetylthio-2-trifluoromethylpropanoic acid

A mixture of thiolactic acid (50 g.) and 2-(trifluoromethyl)acrylic acid[M. W. Buxton, et al., J. Chem. Soc., 366 (1954)] (66 g.) is heated onthe steam bath for one hour and then stored at room temperature foreighteen hours. The reaction mixture is distilled in vacuo to give3-acetylthio-2-trifluoromethylpropanoic acid.

EXAMPLE 31 Synthesis ofN-(3-Acetylthio-2-trifluoromethylpropanoyl)-L-proline tert-butyl ester

L-proline tert-butyl ester (5.1 g.) is dissolved in dichloromethane (40ml.) and the solution is stirred and chilled in an ice bath.Dicyclohexylcarbodiimide (6.2 g.) dissolved in dichloromethane (15 ml.)is added followed immediately by a solution of3-acetylthio-2-trifluoromethylpropanoic acid (6.5 g.) in dichloromethane(5 ml.). After fifteen minutes stirring in the ice bath and sixteenhours at room temperature, the precipitate formed is filtered off andthe filtrate is concentrated to dryness in vacuo. The residue isdissolved in ethyl acetate and washed neutral. The organic phase isdried over magnesium sulfate and concentrated to dryness in vacuo togive N-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline tert-butylester.

EXAMPLE 32 Synthesis ofN-(3-Acetylthio-2-trifluoromethylpropanoyl)-L-proline

N-(3-Acetylthio-2-trifluoromethylpropanoyl)-L-proline tert-butyl ester(8 g.) is dissolved in a mixture of anisole (55 ml.) and trifluoroaceticacid (110 ml.). After one hour storage at room temperature the solventis removed in vacuo and the residue is precipitated several times forether-hexane to giveN-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline.

EXAMPLE 33 Synthesis ofN-(3-Mercapto-2-trifluoromethylpropanoyl)-L-proline

N-(3-Acetylthio-2-trifluoromethylpropanoyl)-L-proline (4 g.) isdissolved in a mixture of water (8 ml.) and concentrated ammonia (8 ml.)under a blanket of nitrogen. After twenty-five minutes stirring at roomtemperature, the reaction mixture is chilled, acidified and extractedwith ethyl acetate. The organic layer is concentrated to dryness invacuo to yield N-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline.

EXAMPLE 34 Synthesis ofN-(2-mercapto-3,3,3-trifluoropropanoyl)-L-proline

To a solution of L-proline (5.75 g.) in 1N sodium hydroxide (50 ml.),chilled in an ice-water bath, 2-bromo-3,3,3-trifluoropropanoic acidchloride (12 g.) is added and the mixture is vigorously stirred at roomtemperature for three hours. A solution of thiolacetic acid (4 ml.) andpotassium carbonate (4.8 g.) in water (50 ml.) is added and the mixtureis stirred at room temperature for sixteen hours. After extraction withethyl acetate, the aqueous layer is acidified with concentratedhydrochloric acid and extracted again with ethyl acetate. This lastorganic phase is dried over magnesium sulfate and concentrated todryness in vacuo. The residue is chromatographed on a silica gel columnwith a mixture of benzene-acetic acid (7:2) to yieldN-(2-acetylthio-3,3,3-trifluoropropanoyl)-L-proline. The named productis obtained by following the procedure of Example 33.

EXAMPLE 35

By substituting the appropriate activated acyl group for the2-bromo-3,3,3-trifluoropropanoic acid chloride in Example 34 or bysubstituting the appropriate thio-acyl group for the3-acetylthio-2-trifluoromethylpropanoic acid in Example 31 and bysubstituting the appropriate amino group for the L--Pro of Example 34 orfor the L-Pro-t-butyl ester of Example 31 and substantially followingthe procedures of Example 34 or Examples 31-33, the mercapto compounds,HS--Z (II), defined in Table 3 are obtained.

                  TABLE 3                                                         ______________________________________                                        R.sub.2      R.sub.3  R.sub.1                                                                              R.sub.1 '                                                                           R.sub.23                                                                           m                                     ______________________________________                                        (1)     --       H        F    F     H    0                                   (2)     --       F        F    F     C.sub.2 H.sub.5                                                                    0                                   (3)     --       CF.sub.3 F    H     H    0                                   (4)     --       C.sub.2 H.sub.5                                                                        F    F     CH.sub.3                                                                           0                                   (5)     --       CH.sub.3 F    H     H    0                                   (6)     --       C.sub.4 H.sub.9                                                                        F    F     H    0                                   (7)     --       CF.sub.3 F    H     C.sub.3 H.sub.7                                                                    0                                   (8)     CF.sub.3 H        H    F     H    1                                   (9)     CF.sub.3 C.sub.3 H.sub.7                                                                        F    F     CH.sub.3                                                                           1                                   (10)    C.sub.2 H.sub.5                                                                        CH.sub.3 F    F     H    1                                   (11)    H        H        F    F     H    1                                   (12)    CF.sub.3 H        H    H     C.sub.4 H.sub.9                                                                    1                                   (13)    C.sub.5 H.sub.11                                                                       CF.sub.3 F    H     H    1                                   (14)    H        CH.sub.3 F    H     H    1                                   (15)    CH.sub.3 C.sub.2 H.sub.5                                                                        F    H     CH.sub.3                                                                           1                                   ______________________________________                                    

Examples 36-40 described the synthesis of HS--Z where Z is defined byformula III. The procedures followed in these examples are described inU.S. Pat. No. 4,154,934.

EXAMPLE 36 Synthesis of N-nitroso-L-proline

To a cooled suspension of 28.2 g. of nitrosyl tetrafluoroborate in 300ml. of dry acetonitrile there is added, with vigorous stirring, over thecourse of 10 minutes, 18.4 g. of L-proline, followed by a solution of 19g. of pyridine in 50 ml. of acetonitrile during 15 minutes. The stirringis continued for an hour and the reaction mixture is then concentratedto dryness under reduced pressure. The residue is extracted with 3×200ml. of ethyl acetate, the ethyl acetate extracts are combined, washedtwice with saturated sodium chloride solution that has been madeslightly acidic with concentrated hydrochloric acid. The ethyl acetatesolution is dried over anhydrous sodium sulfate, filtered andconcentrated to dryness at room temperature under reduced pressure. Theproduct, N-nitroso-L-proline melts at 108°-109° (dec.) aftercrystallization from a mixture of ether and petroleum ether (30°-60°).

EXAMPLE 37 Synthesis of N-amino-L-proline

A solution of 10 g. of N-nitroso-L-proline in 500 ml. of 50% acetic acidis cooled in an ice bath and 40 g. of zinc dust is added gradually, withvigorous stirring, at a rate that the temperature of the reactionmixture is maintained below 10°. The addition requires about 15 minutes.The unreacted zinc dust is removed by filtration and the filtratetreated with hydrogen sulfide to precipitate the zinc as zinc sulfide.The precipitated zinc sulfide is removed by filtration and the filtrateevaporate to dryness. The residue is dissolved in 30 ml. of absoluteethanol and the solution allowed to remain overnight at 5°. TheN-amino-L-proline, a yellow crystalline solid, is removed by filtrationand melts at 153°-154° after drying.

EXAMPLE 38 Synthesis ofN[[3-(Acetylmercapto)-1-oxopropyl]amino]-L-proline

To a suspension of 3.9 g. of N-amino-L-proline and 6.06 g. ofN-methylmorpholine in 200 ml. of dimethylacetamide is added 4.98 g. of3-acetylthiopropionyl chloride. The temperature of the reaction mixturerises to 34° spontaneously. The reaction mixture is then heated at 90°for 5 hours and allowed to cool to room temperature overnight. Thecrystalline solid, N-methylmorpholine hydrochloride, is removed byfiltration and the filtrate concentrated under reduced pressure. Theresidue is dissolved in a minimum amount of 20% hydrochloric acid andthe aqueous solution is then extracted with 3×150 ml. of ethyl acetate.The ethyl acetate extracts are combined, dried over anhydrous magensiumsulfate, and then concentrated under reduced pressure to yield thedesired N-[[3-(acetylmercapto)-1-oxopropyl]amino]-L-proline.

EXAMPLE 39 Synthesis of N-[(3-Mercapto-1 -oxopropyl)amino]-L-proline

Nitrogen is bubbles into a solution of 12 ml. of concentrated aqueousammonia in 25 ml. of water at 10° for 15 minutes. To this solution thereis added 5.8 g. of N-[[(3-acetylmercapto)-1-oxopropyl]amino]-L-prolineand the resulting solution is stirred for 21/2 hours under nitrogen. Itis then cooled in an ice-bath and made strongly acidic with 20%hydrochloric acid. The mixture is extracted with 3×150 ml. of ethylacetate, the ethyl acetate extracts are dried over anhydrous magnesiumsulfate and then concentrated under reduced pressure. The oily residueis triturated with ether, the ether decanted and the last traces ofether removed under reduced pressure. The oily residue is dissolved inwater and lyophilized to yieldN-[(3-mercapto-1-oxopropyl)amino]-L-proline hemihydrate as a viscousoil.

Analysis calcd. for C₈ H₁₄ N₂ O₃ S.1/2H₂ O: C, 42.27; H, 6.65; N, 12.32;S, 14.11 Found: C, 42.59; H, 6.68; N, 12.29; S, 14.29.

EXAMPLE 40

By substituting the appropriate starting materials into Examples 36-39and substantially following the procedures of Examples 36-39, themercapto compounds HS--Z (III), defined in Table 4 are obtained.

                  TABLE 4                                                         ______________________________________                                        R.sub.4       R.sub.5  R.sub.6   R.sub.23                                                                           m   n                                   ______________________________________                                         (1)  --          C.sub.2 H.sub.5                                                                        H       H    0   1                                  (2)  --          CH.sub.3 OH      CH.sub.3                                                                           0   1                                  (3)  --          H        H       C.sub.3 H.sub.7                                                                    0   1                                  (4)  --          H        H       H    0   2                                  (5)  --          CH.sub.3 H, 3-OH H    0   2                                  (6)  --          H        H, 4-OH CH.sub.3                                                                           0   2                                  (7)  --          C.sub.6 H.sub.5 CH.sub.2                                                               H       H    0   2                                  (8)  --          H        H       C.sub.4 H.sub.9                                                                    0   3                                  (9)  --          C.sub.3 H.sub.7                                                                        H, H, 5-OH                                                                            H    0   3                                 (10)  H           H        H       H    1   1                                 (11)  H           H        OH      CH.sub.3                                                                           1   1                                 (12)  C.sub.2 H.sub.5                                                                           CH.sub.3 H       H    1   1                                 (13)  H           CH.sub.3 H, 3-I  H    1   2                                 (14)  H           H        H, 3-OH C.sub.2 H.sub.5                                                                    1   2                                 (15)  C.sub.2 H.sub.5                                                                           C.sub.2 H.sub.5                                                                        H       H    1   2                                 (16)  C.sub.6 H.sub.5 CH.sub.2 CH.sub.2                                                         H        H, 4-OH CH.sub.3                                                                           1   2                                 (17)  CH.sub.3    H        H       H    1   3                                 (18)  H           H        H, H, 3-OH                                                                            C.sub.3 H.sub.7                                                                    1   3                                 (19)  H           C.sub.4 H.sub.9                                                                        H, H, 4-OH                                                                            H    1   3                                 (20)  CH.sub.3    C.sub.2 H.sub.5                                                                        H       H    1   3                                 ______________________________________                                    

Examples 41-44 describe the synthesis of HS--Z where Z is defined byformula X. The procedures followed in these examples are described inU.S. Pat. No. 4,129,566.

EXAMPLE 41 Synthesis ofN-(3-Acetylthio-2-ethylpropanoyl)-L-3,4-dehydroproline

L-3,4-dehydroproline (3.4 g.) is dissolved in 1.0N sodium hydroxide (30ml.) and the solution is chilled in an ice-water bath.3-Acetylthio-2-ethylpropanoyl chloride (5.84 g.) and 2N sodium hydroxide(15 ml.) are added and the solution is stirred at room temperature for 3hours. The mixture is extracted with ether, acidified and extracted withethyl acetate. The organic phase is dried over magnesium sulfate andevaporated to dryness to yieldN-(3-acetylthio-2-ethylpropanoyl)-L-3,4-dehydroproline.

EXAMPLE 42 Synthesis ofN-(2-Ethyl-3-mercaptopropanoyl)-L-3,4-dehydroproline

N-(3-Acetylthio-2-ethylpropanoyl)-L-3,4-dehydroproline (3 g.) isdissolved in a mixture of water (10 ml.) and concentrated ammonia (10ml.) under a blanket of nitrogen. After 25 minutes, the reaction mixtureis acidified and extracted with ethyl acetate. The organic layer isdried over magnesium sulfate and evaporated to dryness to yieldN-(2-ethyl-3-mercaptopropanoyl)-L-3,4-dehydroproline.

EXAMPLE 43 Synthesis ofN-(2-mercaptopropanoyl)-D,L-4,5-dehydropiperidine-2-carboxylic acid

D,L-4,5-dehydropiperidine-2-carboxylic acid (5.65 g.) is dissolved in1.0N aqueous sodium hydroxide (50 ml.) and the solution is chilled in anice-water bath with stirring. 2N Sodium hydroxide (25 ml.) and2-bromopropanoyl chloride (8.57 g.) are added. The mixture is stirred atroom temperature for one hour. A mixture of thioacetic acid (4.18 g.)and potassium carbonate (4.8 g.) in water (50 ml.) is added and themixture is stirred at room temperature for eighteen hours. Afteracidification, the mixture is extracted with ethyl acetate. The organiclayer is dried over magnesium sulfate and concentrated to dryness invacuo to yieldN-(2-acetylthiopropanoyl)-D,L-4,5-dehydropiperidine-2-carboxylic acid.The named product is obtained by following the procedure of Example 42.

EXAMPLE 44

By substituting the appropriate starting materials into Examples 41 to43 and substantially following the procedures of Examples 41-43, themercapto compounds, HS--Z (X), defined in Table 5 are obtained.

                  TABLE 5                                                         ______________________________________                                                 R.sub.22 R.sub.23  t        u                                        ______________________________________                                        (1)        H          C.sub.2 H.sub.5                                                                         0      0                                      (2)        C.sub.3 H.sub.7                                                                          H         0      0                                      (3)        H          H         0      0                                      (4)        CH.sub.3   H         0      1                                      (5)        H          C.sub.4 H.sub.9                                                                         0      1                                      (6)        CH.sub.3   C.sub.3 H.sub.7                                                                         1      0                                      (7)        C.sub.5 H.sub.11                                                                         H         1      0                                      (8)        H          CH.sub.3  1      0                                      (9)        CH.sub.3   H         1      1                                      (10)       C.sub.2 H.sub.5                                                                          H         1      1                                      (11)       CH.sub.3   CH.sub.3  1      1                                      ______________________________________                                    

Examples 45-48 describe the synthesis of HS--Z where Z is defined byformula IX. The procedures followed in these examples are described inU.S. Pat. No. 4,108,886.

EXAMPLE 45 Synthesis of2-[(3-Benzoylthiopropanoyl)amino]-2-methylpropanoic acid

a-Aminoisobutyric acid (5.15 g.) is dissolved in 59 ml. of 0.85N sodiumhydroxide while stirring in an ice bath. To this, 25 ml. of 2N sodiumhydroxide is added, followed by 8.5 g. of 3-bromopropionyl chloride. Thebath is removed, and the pH adjusted to 7.3 with 2N sodium hydroxide.After 2 hours, a solution of 7.5 g. of thiobenzoic acid and 4.8 g. ofpotassium carbonate in 50 ml. of water is added. The reaction mixture isstirred overnight at room temperature, acidified with concentratedhydrochloric acid and extracted with ethyl acetate. The organic layer isdried and concentrated to dryness, yield 13.1 g. The product,2-[(3-benzoylthiopropanoyl)amino]-2-methylpropanoic acid, iscrystallized from ethylacetate-ether, yield 5.4 g., m.p. 142°-143°.

EXAMPLE 46 Synthesis of 2-[(3-Mercaptopropanoyl)amino]-2-methylpropanoicacid

2.8 g. of the product of Example 45 is treated with a mixture of 20 ml.water and 20 ml. of concentrated ammonium hydroxide solution under anargon blanket for one hours. The benzamide precipitate is filtered andthe filtrate is extracted twice with ethyl acetate. The aqueous phase isconcentrated in vacuo, acidified with concentrated hydrochloric acid andextracted with ethyl acetate. The organic layer is dried andconcentrated to dryness in vacuo and the residual product2-[(3-mercaptopropanoyl)amino]-2-methylpropanoic acid, is crystallizedfrom acetonitrile, yield 1.2 g., m.p. 169°-170°.

EXAMPLE 47 Synthesis of 1-[(3-mercaptopropanoyl)amino]cyclopentanecarboxylic acid

1-Aminocyclopentane-1-carboxylic acid (6.45 g.) is dissolved in 50 ml.of 1N sodium hydroxide solution and stirred in an ice bath. To this 25ml. of 2N sodium hydroxide solution is added, followed immediately with8.5 g. of 3-bromopropionyl chloride. The bath is removed and the pH isabout 7. Some crystals come out of solution. After 3.5 hours at roomtemperature, 54 ml. of 1N sodium hydroxide solution is added andeverything goes into solution. This is followed immediately with 4.12 g.of thiolacetic acid. An additional 5 ml. of 1N sodium hydroxide is addedto bring the pH to near 8. After standing overnight, the mixture isacidified with concentrated hydrochloric acid, extracted with ethylacetate, dried over magnesium sulfate, and concentrated to dryness invacuo. The product, 1-[(3-acetylthiopropanoyl)amino]cyclopentanecarboxylic acid, is first crystallized from ethyl acetate and hexane.This material is recrystallized from ethyl acetate, yield 3.655 g., m.p.127°-128°. The named product is obtained by following the procedure ofExample 46.

EXAMPLE 48

By substituting the appropriate starting materials into Example 45 andsubstantially following the procedures of Example 45 and 46, themercapto compounds, HS--Z (IX), defined in Table 6 are obtained.

                  TABLE 6                                                         ______________________________________                                                            methylene bridge                                          R.sub.18  R.sub.19                                                                              R.sub.20                                                                              R.sub.21                                                                            R.sub.19 -R.sub.20                                                                    R.sub.19 -R.sub.21                    ______________________________________                                        (1)  CH.sub.3 C.sub.2 H.sub.5                                                                       CH.sub.3                                                                            H     --      --                                  (2)  H        CH.sub.3                                                                              C.sub.4 H.sub.9                                                                     CH.sub.3                                                                            --      --                                  (3)  C.sub.3 H.sub.7                                                                        CH.sub.3                                                                              C.sub.2 H.sub.5                                                                     C.sub.2 H.sub.5                                                                     --      --                                  (4)  CH.sub.3 --      --    H     (CH.sub.2).sub.4                                                                      --                                  (5)  H        --      --    C.sub.3 H.sub.7                                                                     (CH.sub.2).sub.4                                                                      --                                  (6)  C.sub.5 H.sub.11                                                                       --      --    CH.sub.3                                                                            (CH.sub.2).sub.4                                                                      --                                  (7)  CH.sub.3 --      H     --    --      (CH.sub.2).sub.3                    (8)  C.sub.4 H.sub.9                                                                        --      H     --    --      (CH.sub.2).sub.3                    (9)  H        --      CH.sub.3                                                                            --    --      (CH.sub.2).sub.3                    (10) C.sub.2 H.sub.5                                                                        --      C.sub.3 H.sub.7                                                                     --    --      (CH.sub.2).sub.3                    ______________________________________                                    

Examples 49-52 describe the synthesis of HS--Z where Z is defined byformula VII. The procedures followed in these examples are described inU.S. Pat. No. 4,053,651.

EXAMPLE 49 Synthesis of N-(3-Benzoylthiopropanoyl)-L-alanine

L-alanine (4.45 g.) is dissolved in aqueous 1.0N sodium hydroxide (50ml.) and the solution is chilled in the ice bath with stirring. 2Nsodium hydroxide (27 ml.) and 3-bromopropionyl chloride (8.5 g.) areadded in that order and the mixture is removed from the ice bath andstirred at room temperature for three and one half hours. A mixture ofthiobenzoic acid (7.5 g.) and potassium carbonate (4.8 g.) in water (50ml.) is added and the mixture is stirred at room temperature overnight.After acidification with concentrated hydrochloric acid the aqueoussolution is extracted with ethyl acetate, and the organic phase iswashed with water, dried and concentrated to dryness. The residue (14.9)is crystallized from ether to yield 7.1 g. ofN-(3-benzoylthiopropanoyl)-L-alanine, m.p. 99°-100°.

EXAMPLE 50 Synthesis of N-(3-mercaptopropanoyl)-L-alanine

N-(3-benzoylthiopropanoyl)-L-analine (4.2 g.) is dissolved in a mixtureof water (7.5 ml.) and concentrated ammonium hydroxide (6 ml.). Afterone hour, the mixture is diluted with water, filtered and the filtrateis extracted with ethyl acetate. The aqueous phase is acidified withconcentrated hydrochloric acid and extracted with ethyl acetate. Theorganic phase is washed with water, dried and concentrated to dryness invacuo. The residue is crystallized from ethyl acetate-hexane to yield1.87 g. of N-(3-mercaptopropanoyl)-L-alanine, m.p. 79°-81°.

EXAMPLE 51 Synthesis of N-(3-mercapto-2-methylpropanoyl)-L-valine

L-valine (88 g.) and sodium carbonate (40 g.) are dissolved in water(11.) and the solution is chilled in an ice bath with vigorous stirring.3-Acetylthio-2-methylpropanoyl chloride (135 g.) and a solution ofsodium carbonate (120 g.) in 500 ml. of water are added in five equalportions over a 15 minute period. After 1.5 hours the reaction mixtureis extracted with ethyl acetate, the aqueous phase is acidified withconcentrated hydrochloric acid and extracted with ethyl acetate. Theorganic phase is washed with water, dried over magnesium sulfate andconcentrated to dryness to yield 190 g. ofN-(3-acetylthio-2-methylpropanoyl)-L-valine. The named product isobtained by following the procedure of Example 50.

EXAMPLE 52

By substituting the appropriate starting materials into Examples 49 or51 and substantially following the procedures of Examples 49-51, themercapto compounds, HS--Z (VII), defined in Table 7 are obtained.

                                      TABLE 7                                     __________________________________________________________________________    R.sub.4     R.sub.5                                                                            R.sub.13                                                                           R.sub.14       q                                        __________________________________________________________________________    (1)                                                                              --       H    H    (CH.sub.2).sub.4NH.sub.2                                                                     0                                        (2)                                                                              --       H    H                                                                                   ##STR20##     0                                        (3)                                                                              --       CH.sub.3                                                                           H    CH.sub.2 CO.sub.2 H                                                                          0                                        (4)                                                                              --       H    CH.sub.3                                                                           CH.sub.2 CH(CH.sub.3).sub.2                                                                  0                                        (5)                                                                              --       C.sub.2 H.sub.5                                                                    CH.sub.3                                                                           C.sub.6 H.sub.5 CH.sub.2                                                                     0                                        (6)                                                                              H        C.sub.6 H.sub.5 CH.sub.2                                                           H    H              1                                        (7)                                                                              CH.sub.3 H    H    CH.sub.2 OH    1                                        (8)                                                                              C.sub.4 H.sub.9                                                                        CH.sub.3                                                                           H                                                                                   ##STR21##     1                                        (9)                                                                              H        H    C.sub.2 H.sub.5                                                                    CH.sub.3SCH.sub.2 CH.sub.2                                                                   1                                        (10)                                                                             CH.sub.3, H                                                                            H    CH.sub.3                                                                           H              2                                        (11)                                                                             H, C.sub.6 H.sub.5 CH.sub.2 CH.sub.2                                                   CH.sub.3                                                                           H                                                                                   ##STR22##     2                                        (12)                                                                             H        C.sub.5 H.sub.11                                                                   H                                                                                   ##STR23##     2                                        (13)                                                                             H        H    H    CH.sub.2 SH    2                                        (14)                                                                             H, CH.sub.3                                                                            H    C.sub.3 H.sub.7                                                                    CH.sub.3       2                                        (15)                                                                             H        C.sub.3 H.sub.7                                                                    H                                                                                   ##STR24##     1                                        (16)                                                                             CH.sub.3 C.sub.2 H.sub.5                                                                    C.sub.6 H.sub.5 CH.sub.2                                                           CH(CH.sub.3)(C.sub.2 H.sub.5)                                                                1                                        __________________________________________________________________________

Examples 53-68 describe the synthesis of HS--Z where Z is defined byformula IV. The procedures followed in these examples are described inU.S. Pat. Nos. 4,113,715 and 4,146,611.

EXAMPLE 53 Synthesis of N,S-Diacetyl-D,L-cysteinyl-L-proline tert-butylester

To a solution of L-proline tert-butyl ester (0.85 g.) andhydroxybenzotriazole (0.67 g.) in methylene chloride (10 ml.) chilled inan ice bath, dicyclohexylcarbodiimide (1.03 g.) andN,S-diacetyl-D,L-cysteine (1.7 g.) are added in that order. Afterfifteen minutes, the ice bath is removed and the mixture is stirred atroom temperature overnight. The precipitate is filtered off and thefiltrate is washed with 10% potassium bisulfate, water, saturated sodiumbicarbonate, and water. The organic phase is dried and concentrated todryness in vacuo to give N,S-diacetyl-D,L-cysteinyl-L-proline tert-butylester as an oil. R_(f) =0.25 (silica gel, ethylacetate).

EXAMPLE 54 Synthesis of N,S-Diacetyl-D,L-cysteinyl-L-proline

N,S-Diacetyl-D,L-cysteinyl-L-proline tert-butyl ester (1.9 g.) isdissolved in a mixture of anisole (6 ml.) and trifluoroacetic acid (12ml.) and the solution is stored at room temperature for one hour. Thesolvents are removed in vacuo and the residue is precipitated from ethylacetate-ether-hexane, to obtain N,S-diacetyl-D,L-cysteinyl-L-proline,yield 1.08 g., m.p. 80°-140°.

EXAMPLE 55 Synthesis of N-Acetyl-D,L-cysteinyl-L-proline

N,S-Diacetyl-D,L-cysteinyl-L-proline (0.3 g.) is dissolved in a mixtureof water (4 ml.) and concentrated ammonia (4 ml.) under a blanket ofargon. The solution is stored for thirty minutes at room temperature,saturated with sodium chloride and extracted with ethyl acetate andchloroform. The organic layers are pooled and concentrated to dryness invacuo to obtain N-acetyl-D,L-cysteinyl-L-proline, yield 0.1 g., R_(f)=0.25 (silica gel; benzene:acetic acid, 75:25).

EXAMPLE 56 Synthesis of Nα-acetyl-3-acetylthiovalyl-L-proline t-butylester

By substituting N,S-diacetyl-penicillamine for theN,S-diacetyle-D,L-cysteine in the procedure of Example 53,Nα-acetyl-3-acetylthiovalyl-L-proline t-butyl ester is obtained.

EXAMPLE 57 Synthesis of Nα-acetyl-3-mercaptovalyl-L-proline

By substituting the product of Example 56 for theN,S-diacetyl-D,L-cysteinyl-L-proline t-butyl ester in the procedure ofExamples 54 and 55, the named product is obtained.

EXAMPLE 58 Synthesis of Methyl N-(p-methoxybenzyl)nipecotatehydrochloride

A mixture of 23 g. of methyl nipecotate, 24.3 g. of potassium carbonate,and 52 g. of p-methoxybenzyl trichloroacetate in 800 ml. of toluene isrefluxed under nitrogen for seventy-two hours. The mixture is cooled,the toluene removed in vacuo, the residue dissolved in chloroform, andthis solution washed once with 400 ml. of aqueous potassium carbonateand then with 400 ml. of 10% hydrochloric acid. The chloroform solutionis dried and concentrated in vacuo to a viscous brown oil. Triturationof this oil with ethyl acetate affords 30.7 g. of methylN-(p-methoxybenzyl)nipecotate hydrochloride as an off-white crystallinesolid. Recrystallization from ethyl acetate yields the analyticalsample, m.p. 150°-154°.

EXAMPLE 59 Synthesis of N-(p-Methoxybenzyl)-3-methylene-2-piperidone

A solution of methyl N-(p-methoxybenzoyl)nipecotate hydrochloride (30.7g.) and 8.4 g. of sodium hydroxide in 900 ml. of methanol and 45 ml. ofwater is stirred at room temperature for seventeen hours. The solutionis evaporated to dryness in vacuo, the residue diluted with toluene, andthis again evaporated to dryness in vacuo. To the residue is added 1liter of acetic anhydride and 140 ml. of triethylamine, and theresulting mixture is heated under reflux for four hours. The reactionmixture is evaporated to dryness in vacuo, the residue taken up inchloroform, washed with water, dried and concentrated in vacuo. Theresidual oil is chromatographed on silica gel using 1:1 hexane-ethylacetate as the eluant, and yields 16.9 g. ofN-(p-methoxybenzyl)-3-methylene-2-piperidone as a chromatographicallypure yellow oil. Alternatively, the oil can be distilled to giveanalytically pure N-(p-methoxybenzyl)-3-methylene-2-piperidone, b.p.145°-155°/0.05 mm.

EXAMPLE 60 Synthesis of 3-Methylene-2-piperidone

A solution of N-(p-methoxybenzyl)-3-methylene-2-piperidone (16.9 g.) and21.3 g. of anisole in 400 ml. of trifluoroacetic acid is refluxed undernitrogen for forty-eight hours. The solution is evaporated to dryness invacuo, and the residue chromatographed on 900 g. of silica gel usingethyl acetate as eluant, yielding 6.5 g. of 3-methylene-2-piperidone asa crystalline solid.

EXAMPLE 61 Synthesis of 2-Methylene-5-aminopentanoic acid hydrochloride

A solution of 2.6 g. of 3-methylene-2-piperidone in 150 ml. of 6Nhydrochloric acid is refluxed for twenty-four hours. The cooled solutionis extracted with chloroform, and the aqueous layer concentrated invacuo to 3.8 g. of glassy foam. The foam is heated with methanol,filtered through Celite (diatomaceous earth clarifying agent) to removea small amount of insoluble material, and the filtrate is evaporated todryness in vacuo, yielding 2.5 g. of 2-methylene-5-aminopentanoic acidhydrochloride as a tan crystalline solid. Recrystallization fromisopropanol gives the analytical sample, m.p. 138°-144°.

EXAMPLE 62 Synthesis of2-Methylene-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid

To a solution of 8.8 g. of 2-methylene-5-aminopentanoic acidhydrochloride in 100 ml. of water is added with stirring 6.36 g. ofmagnesium oxide, followed by a solution of 12.2 g. ofp-methoxybenzyloxycarbonyl azide in 100 ml. of dioxane, and theresulting mixture is stirred at room temperature for two days. Thereaction mixture is filtered, and the filtrate diluted with 200 ml. ofethyl acetate, two equivalents of Dowex 50 ion exchange resin is added,and the mixture is stirred at room temperature for two hours. The resinis then filtered off and washed with water. The layers in the filtrateare separated and the aqueous layer is extracted twice with ethylacetate. The combined organic layers are dried and concentrated in vacuoto give 18.2 g. of2-methylene-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid as anamber oil which crystallizes on standing. This is used without furtherpurification.

EXAMPLE 63 Synthesis of2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)amino pentanoic acid

A solution of 2-methylene-5-(p-methoxybenzyloxycarbonyl)amino pentanoicacid (53 mmoles) in 50 ml. of thiolacetic acid is allowed to stand atroom temperature for forty-eight hours. The solution is evaporated todryness in vacuo, and the residue taken up in chloroform and applied toa silica gel column (700 g.). Elution with 5% methanol in chloroformaffords 14.2 g. of2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid asan oil. Treatment of this oil with one equivalent of dicyclohexylaminein ether, followed by recrystallization from ethyl acetate affords thecorresponding dicyclohexylamine salt, m.p. 112°-114°.

EXAMPLE 64 Synthesis of2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)amino pentanoic acidN-hydroxysuccinimide ester

To a solution of 3.7 g. of2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid and1.21 g. of N-hydroxysuccinimide in 60 ml. of dichloromethane at 0°-5° isadded 2.16 g. of N,N'-dicyclohexylcarbodiimide over twenty minutes withstirring. The resulting mixture is stirred overnight at 0°-5°. Theprecipitated dicyclohexylurea is filtered off, the filtrate concentratedin vacuo and the residue taken up in ethyl acetate and washed through asilica gel column to give 4.6 g. of2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)amino pentanoic acidN-hydroxysuccinimide ester as an oil, which crystallizes on triturationwith ether. Recrystallization from ethyl acetate-hexane affords theanalytical sample, m.p. 85°-87°.

EXAMPLE 65 Synthesis ofN-[(2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl)]-L-prolinetert-butyl ester

By substituting2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoic acid forthe N,S-diacetyl-D,L-cysteine in the procedure of Example 53,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-prolinetert-butyl ester is obtained.

EXAMPLE 66 Synthesis ofN-(2-Acetylthiomethyl-5-aminopentanoyl)-L-proline, trifluoroacetate salt

N-[2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-prolinetert-butyl ester (2 g.) is dissolved in a mixture of trifluoroaceticacid (15 ml.) and anisole (6 ml.). The solution is stored at roomtemperature for one hour, the solvents are removed in vacuo and theresidue is precipitated from ethyl acetate-ether to yieldN-(2-acetylthiomethyl-5-aminopentanoyl)-L-proline, trifluoroacetate.

EXAMPLE 67 Synthesis of N-(5-Amino-2-mercaptomethylpentanoyl)-L-proline

N-(2-Acetylthiomethyl-5-aminopentanoyl)-L-proline trifluoroacetate (1g.) is dissolved in a mixture of water (12 ml.) and concentrated ammonia(12 ml.) under a blanket of argon. The solution is stored twenty minutesat room temperature concentrated to 5 ml. and applied to a column ofDowex 50 ion exchange resin in the hydrogen cycle. The column is washedwith water and N-(5-amino-2-mercaptomethylpentanoyl)-L-proline is elutedwith a buffer of pyridine-acetic acid at pH 6.5.

EXAMPLE 68

By substituting the appropriate starting materials into Examples 53-67and substantially following the procedures of Examples 53-67, themercapto compounds, HS--Z (IV), defined in Table 8 are obtained.

                                      TABLE 8                                     __________________________________________________________________________                                       methylene bridge                           R.sub.4 R.sub.7                                                                             R.sub.8                                                                             R.sub.9        R.sub.8 -R.sub.9                                                                       R.sub.23                                                                         m p                            __________________________________________________________________________    (1)                                                                              H    H     H     CH.sub.3       --       H  1 0                            (2)                                                                              CH.sub.3                                                                           H     H     H              --       H  1 0                            (3)                                                                              H                                                                                   ##STR25##                                                                          CH.sub.3                                                                            CH.sub.2 OH    --       CH.sub.3                                                                         1 0                            (4)                                                                              C.sub.3 H.sub.7                                                                    H     H     CH.sub.2 CH.sub.2 CO.sub.2 H                                                                 --       H  0 1                            (5)                                                                              H                                                                                   ##STR26##                                                                          --    --             (CH.sub.2).sub.3                                                                       H  0 1                            (6)                                                                              H    H     H                                                                                    ##STR27##     --       C.sub.2 H.sub.5                                                                  0 1                            (7)                                                                              H                                                                                   ##STR28##                                                                          --    --             (CH.sub.2).sub.4                                                                       H  1 1                            (8)                                                                              C.sub.2 H.sub.5                                                                     ##STR29##                                                                          --    --             (CH.sub.2).sub.3, 3-OH                                                                 C.sub.4 H.sub.9                                                                  1 1                            (9)                                                                              CH.sub.3                                                                           H     H     (CH.sub.2).sub.4 NH.sub.2                                                                    --       H  1 1                            (10)                                                                             H    H     --    --             (CH.sub.2).sub.4, 5-OH                                                                 H  0 2                            (11)                                                                             H                                                                                   ##STR30##                                                                          C.sub.2 H.sub.5                                                                      ##STR31##     --       H  0 2                            (12)                                                                             CH.sub.3                                                                           H     H     CH(CH.sub.3).sub.2                                                                           --       H  0 2                            (13)                                                                             C.sub.6 H.sub.5 CH.sub.2                                                           H     H     CH.sub.2 CH(CH.sub.3).sub.2                                                                  --       H  1 2                            (14)                                                                             H                                                                                   ##STR32##                                                                          --    --             (CH.sub.2).sub.3, 4-F                                                                  CH.sub.3                                                                         1 2                            (15)                                                                             H    H     H     CH.sub.2 CH.sub.2 SCH.sub.3                                                                  --       H  0 3                            (16)                                                                             CH.sub.3                                                                            ##STR33##                                                                          CH.sub.3                                                                             ##STR34##     --       H  0 3                            (17)                                                                             H    H     H     C.sub.6 H.sub.5 CH.sub.2                                                                     --       H  1 3                            (18)                                                                             H                                                                                   ##STR35##                                                                          H     CH.sub.2 SH    --       H  1 3                            (19)                                                                             CH.sub.3                                                                           H     --    --             (CH.sub.2).sub.3                                                                       H  0 4                            (20)                                                                             H                                                                                   ##STR36##                                                                          H                                                                                    ##STR37##     --       CH.sub.3                                                                         0 4                            (21)                                                                             H    H     H                                                                                    ##STR38##     --       H  1 4                            (22)                                                                             H                                                                                   ##STR39##                                                                          CH.sub.2 OH                                                                         CH.sub.3       --       H  1 4                            __________________________________________________________________________

Examples 69-78 describe the synthesis of HS--Z where Z is defined byformula V. The procedures followed in these examples are described inU.S. Pat. No. 4,116,962.

EXAMPLE 69 Synthesis of 2-(Acetylthiomethyl)-3-(acetylthio)propanoicacid

A solution of 3.36 g. (40 mmoles) of thiolacetic acid in 40 ml. ofN-potassium hydroxide is added dropwise to a solution of2-bromomethyl-3-bromopropanoic acid in 1.0N potassium hydroxide (20ml.). The mixture is stirred at room temperature overnight, acidifiedwith concentrated hydrochloric acid and extracted with ethyl acetate.The organic layer is dried and concentrated in vacuo. The residue isconverted into a dicyclohexylammonium salt (m.p. 116°-118°) and the saltconverted back into the free acid,2-(acetylthiomethyl)-3-(acetylthio)propanoic acid, by distributionbetween ethyl acetate and 10% potassium bisulfate.

EXAMPLE 70 Synthesis ofN-[(2-Acetylthiomethyl)-3-(acetylthio)propanoyl]-L-proline

To a solution of L-proline (1.44 g.) and sodium carbonate (2.7 g.) inwater (25 ml.) in an ice bath,2-(acetylthiomethyl)-3-(acetylthio)propanic acid chloride (3.9g.--prepared from 2-(acetylthiomethyl)-3-(acethythio)-propanoic acid andthionyl chloride) is added and the mixture is vigorously stirred at roomtemperature for two hours. After extraction with ethyl acetate, theaqueous layer is acidified and extracted with ethyl acetate. The organiclayer is dried and concentrated to dryness. The residue ischromatographed on a column of silica gel with a mixture ofbenzene-acetic acid (7:1). The fractions containing the desired materialare pooled and concentrated to dryness to yieldN-[(2-acetylthiomethyl)-3-(acethylthio)-propanoyl]-L-proline as an oil(1.3 g.) R_(f) : 0.3 (silica gel:benzene-acetic acid, 75:25).

EXAMPLE 71 Synthesis ofN-(2-Mercaptomethyl-3-mercaptopropanoyl)-L-proline

N-[(2-Acetylthiomethyl-3-(acetylthio)propanoyl]-L-proline (1.2 g.) isdissolved in a mixture of water (12 ml.) and concentrated ammonia (12ml.) under an atmosphere of argon. After twenty minutes, the mixture isacidified with concentrated hydrochloric acid. The crystallineprecipitate N-(2-mercaptomethyl-3-mercaptopropanoyl)-L-proline isfiltered and dried, yield 0.63 g., m.p. 138°-140°.

EXAMPLE 72 Synthesis of 3-(Acetylthio)-2-(methylthiomethyl)propanoicacid

A mixture of 3-(methylthiomethyl)acrylic acid (5.5 g.) and thiolaceticacid (5 ml.) is heated in the steam bath until disappearance of vinylproton absorption in the nmr. The mixture is concentrated to remove theexcess thiolacetic acid to obtain3-(acetylthio)-2-(methylthiomethyl)propanoic acid.

EXAMPLE 73 Synthesis ofN-(3-Acetylthio)-2-methylthiomethyl)propanoyl]-L-proline

To a solution of L-proline (1.44 g.) and sodium carbonate (2.7 g.) inwater (25 ml.) in an ice bath,3-(acetylthio)-2-(methylthiomethyl)propanoic acid chloride (preparedfrom the acid of Example 72 with thionyl chloride) (3.6 g.) is added,and the mixture is vigorously stirred at room temperature for two hours.After extraction with ethyl acetate, the aqueous layer is acidified andextracted with ethyl acetate. The organic layer is dried andconcentrated to dryness in vacuo to giveN-[3-(acetylthio)-2-methylthiomethyl)propanoyl]-L-proline.

EXAMPLE 74 Synthesis ofN-[3-Mercapto-2-(methylthiomethyl)propanoyl]-L-proline

N-[3-(acetylthio)-2-(methylthiomethyl)propanoyl]-L-proline (1.2 g.) isdissolved in a mixture of water (12 ml.) and concentrated ammonia (12ml.) under a blanket of argon. After twenty minutes, the reactionmixture is acidified and extracted with ethyl acetate. The organic layeris dried and concentrated to dryness to yieldN-([3-mercapto-2-methylthiomethyl)propanoyl]-L-proline.

EXAMPLE 75 Synthesis ofN-(2-Hydroxymethyl-3-mercaptopropanoyl)-L-proline

N-[2-acetoxymethyl-3-(acetylthio)propanoyl]-L-proline (1.5 g.) isdissolved in a mixture of water (12 ml.) and concentrated ammonia (12ml.) under a blanket of argon. After one hour, the reaction mixture isconcentrated to ca. dryness, diluted with water and the solution appliedto a column of cation exchange resin (Dowex 50) in the hydrogen cycle.The water eluate is concentrated to small volume and freeze dried toyield N-(2-hydroxymethyl-3-mercaptopropanoyl)-L-proline.

EXAMPLE 76 Synthesis of N-[2-Benzoylthio-3-methoxybutanoyl]-L-proline

To a solution of L-proline (5.75 g.) in N sodium hydroxide (50 ml.)chilled in an ice bath, 2N sodium hydroxide (25 ml.) and2-bromo-3-methoxybutyric acid chloride [obtained from2-bromo-3-methoxybutyric acid [J. Am. Chem. Soc., 71, 1096, (1949)] andthionyl chloride] (10.7 g.) are added, with vigorous stirring. Afterthree hours, thiobenzoic acid (7.5 g.) and potassium carbonate (4.8 g.)are added and the mixture is stirred at room temperature overnight. Thereaction mixture is acidified and extracted with ethyl acetate. Theorganic layer is concentrated to dryness and the residue ischromatographed on a column of silica gel with benzene-acetic acid toyield N-[2-benzoylthio-3-methoxybutanoyl]-L-proline.

EXAMPLE 77 Synthesis of N-[2-Mercapto-3-methoxybutanoyl]-L-proline

By substituting N-[2-benzoylthio-3-methoxybutanoyl]L-proline for theN-[3-acetylthio-2-(methylthiomethyl)propanoyl]-L-proline in theprocedure of Example 74, N-[2-mercapto-3-methoxybutanoyl]-L-proline isobtained.

EXAMPLE 78

By substituting the appropriate starting materials into Examples 69-77and substantially following the procedures of Examples 69-77, themercapto compounds, HS--Z (V), defined in Table 9 are obtained.

                                      TABLE 9                                     __________________________________________________________________________                                       methylene bridge                           X    R.sub.10                                                                            R.sub.11                                                                           R.sub.8                                                                            R.sub.9       R.sub.8 -R.sub.9                                                                       R.sub.23                                                                         m p                            __________________________________________________________________________    (1)                                                                              S --    C.sub.2 H.sub.5                                                                    H    H             --       H  0 0                            (2)                                                                              O --    H    --   --            (CH.sub.2).sub.3                                                                       CH.sub.3                                                                         0 0                            (3)                                                                              S --                                                                                   ##STR40##                                                                         H    CH.sub.2 CO.sub.2 H                                                                         --       H  1 0                            (4)                                                                              O --    H    --   --            (CH.sub.2).sub.3, 3-Cl                                                                 H  1 0                            (5)                                                                              S C.sub.2 H.sub.5                                                                     H    CH.sub.3                                                                           CH(OH)CH.sub.3                                                                              --       H  0 1                            (6)                                                                              S H     CH.sub.3                                                                           H                                                                                   ##STR41##    --       H  1 1                            (7)                                                                              O H, CH.sub.3                                                                         C.sub.4 H.sub.9                                                                    --   --            (CH.sub.2).sub.4                                                                       C.sub.2 H.sub.5                                                                  0 2                            (8)                                                                              S H                                                                                    ##STR42##                                                                         H    CH.sub.2 SH   --       H  0 2                            (9)                                                                              S H     H    CH.sub.2 OH                                                                        (CH.sub.2).sub.4 NH.sub.2                                                                   --       H  1 2                            (10)                                                                             O CH.sub.3, H                                                                         C.sub.3 H.sub.7                                                                    --   --            (CH.sub.2).sub.3, 3-OH                                                                 C.sub.4 H.sub.9                                                                  1 2                            (11)                                                                             S H                                                                                    ##STR43##                                                                         H                                                                                   ##STR44##    --       H  0 3                            (12)                                                                             S H, H, C.sub.3 H.sub.7                                                               H    H    CH(CH.sub.3).sub.2                                                                          --       H  0 3                            (13)                                                                             S H                                                                                    ##STR45##                                                                         --   --            (CH.sub.2).sub.4, 5-OH                                                                 H  1 3                            (14)                                                                             O H     H    H    CH.sub.3 SCH.sub.2                                                                          --       CH.sub.3                                                                         1 3                            (15)                                                                             O H     CH.sub.3                                                                           C.sub.3 H.sub.7                                                                    CH.sub.2 CH.sub.2 SCH.sub.3                                                                 --       H  0 4                            (16)                                                                             O H     H    H    C.sub.6 H.sub.5 CH.sub.2                                                                    (CH.sub.3).sub.2                                                                       H  0 4                            (17)                                                                             O H     H    H    CH(CH.sub.3)(C.sub.2 H.sub.5)                                                               --       H  1 4                            (18)                                                                             S H                                                                                    ##STR46##                                                                         H    CH.sub.3      --       H  1 4                            __________________________________________________________________________

Examples 79-86 describe the synthesis of HS--Z where Z is defined byformula VI. The procedures followed in these examples are described inU.S. Pat. No. 4,091,024.

EXAMPLE 79 Synthesis of 3-acetylthio-2-methoxycarbonylmethyl propanoicacid

A mixture of thiolacetic acid (12.5 g.) and3-methoxycarbonyl-2-methylenepropanoic acid (17.1 g.) are heated on asteam bath for two hours. The reaction is concentrated in vacuo and theresidue is dissolved in ethyl acetate (125 ml.) and dicyclohexylamine(35 ml.) is added. The crystals are filtered, dried and recrystallizedfrom ethyl acetate to yield 37.8 g., m.p. 120°-121°. Thisdicyclohexylammonium salt of3-acetylthio-2-methoxycarbonylmethylpropanoic acid is converted to thefree acid by distribution between a system of ethyl acetate and 10%aqueous potassium bisulfate.

EXAMPLE 80 Synthesis ofN-[3-(acetylthio)-2-(methoxycarbonylmethyl)propanoyl]-L-proline t-butylester

To a solution of L-proline t-butyl ester (1.71 g.) and3-hydroxybenzotriazole (1.35 g.) in dichloromethane (15 ml.),dicyclohexylcarbodiimide (2.06 g.) and the product from Example 79 (2.2g.) are added. After 18 hours stirring at room temperature, theprecipitate formed is filtered off, the filtrate is washed neutral,dried and concentrated to dryness to yield 3.7 g. of the named productR_(f) : 0.8 (silica gel-ethyl acetate).

EXAMPLE 81 Synthesis ofN-[3-(acetylthio)-2-(methoxycarbonylmethyl)propanoyl]-L-proline

2.9 g. of the product from Example 80 is dissolved in a mixture oftrifluoroacetic acid (17.5 ml.) and anisole (8.4 ml.). After one hourstorage at room temperature, the excess trifluoroacetic acid is removedin vacuo and the residue is precipitated twice from ether-hexane toyield 2.1 g. of the named product. R_(f) : 0.4 (silicagel-benzene:acetic acid, 75:25).

EXAMPLE 82 Synthesis ofN-[3-Mercapto-2-(methoxycarbonylmethyl)propanoyl]-L-proline

2.1 g. of the product from Example 81 is dissolved in a mixture of water(35 ml.) and concentrated ammonia (35 ml.) under a blanket of argon.After 20 minutes, the solution is chilled in an ice bath, made acidicwith concentrated hydrochloric acid, saturated with sodium chloride andextracted with ethyl acetate. The organic layer is dried andconcentrated to dryness in vacuo to yield 1.1 g. of the named productthat is purified by chromatography on silica gel (benzene:acetic acid75:25) R_(f) : 0.35 (silica gel-benzene:acetic acid, 75:25).

EXAMPLE 83 Synthesis ofN-[2-Carboxymethyl-3-mercaptopropanoyl]-L-proline

To a solution of the product from Example 81 (3.0 g.) in methanol (60ml.), 1N sodium hydroxide (60 ml.) is added. After 4 hours, the solutionis applied to a column of Dowex 50 ion exchange resin in the hydrogencycle, and the desired material is eluted with water to yield 2.3 g. ofthe named product R_(f) 0.2 (silica gel-benzene:acetic acid, 75:25).

EXAMPLE 84 Synthesis ofN-[2-carbamoylmethyl-3-mercaptopropanoyl]-L-proline

2.1 g. of the product from Example 81 is dissolved in a mixture of water(40 ml.) and concentrated ammonia (40 ml.). After one hour the reactionmixture is concentrated to 1/3 volume, and applied to a column of Dowex50 resin in the hydrogen cycle. The product is eluted with water. Theaqueous is extracted with ethyl acetate and then concentrated to drynessto yield 1.4 g. of the named product R_(f) 0.50 (silicagel-Chloroform:methanol:acetic acid:water).

EXAMPLE 85 Synthesis ofN-[2-([N-butylcarbamoyl]methyl)-3-mercaptopropanoyl]-L-proline

By substituting 3-(acetylthio)-2-[(N-butylcarbamoyl)methyl]propanoicacid for the 3-(acetylthio)-2-(methoxycarbonylmethyl)propanoic acid inExample 80 and substantially following the procedures of Examples 80-82,the named product is obtained.

EXAMPLE 86

By substituting the appropriate starting materials into Examples 79-85and substantially following the procedures of Examples 79-85, themercapto compounds, HS--Z (VI), defined in Table 10 are obtained.

                                      TABLE 10                                    __________________________________________________________________________                                methylene bridge                                  R.sub.12   R.sub.8                                                                          R.sub.9       R.sub.8 -R.sub.9                                                                       R.sub.23                                                                         m p                                   __________________________________________________________________________    (1)                                                                              CN      H  CH.sub.3      --       H  0 0                                   (2)                                                                              CO.sub.2 H                                                                            H  H             --       H  0 0                                   (3)                                                                               ##STR47##                                                                            C.sub.2 H.sub.5                                                                  CH(CH.sub.3).sub.2                                                                          --       CH.sub.3                                                                         1 0                                   (4)                                                                               ##STR48##                                                                            -- --            (CH.sub.2).sub.4                                                                       H  1 0                                   (5)                                                                               ##STR49##                                                                            H  CH.sub.2 OH   --       C.sub.4 H.sub.9                                                                  0 1                                   (6)                                                                              CO.sub.2 H                                                                            H  (CH.sub.2).sub.4 NH.sub.2                                                                   --       H  0 1                                   (7)                                                                               ##STR50##                                                                            -- --            (CH.sub.2).sub.3, 3-OH                                                                 H  0 2                                   (8)                                                                               ##STR51##                                                                            H                                                                                 ##STR52##    --       H  0 2                                   (9)                                                                               ##STR53##                                                                            C.sub.3 H.sub.7                                                                  CH.sub.2SH    --       H  1 2                                   (10)                                                                             CN      H                                                                                 ##STR54##    --       C.sub.2 H.sub.5                                                                  1 2                                   (11)                                                                             CO.sub.2 H                                                                            -- --            (CH.sub.2).sub.4, 4-OH                                                                 H  0 3                                   (12)                                                                              ##STR55##                                                                            H  CH.sub.2 CO.sub.2 H                                                                         --       H  0 3                                   (13)                                                                             CN      H                                                                                 ##STR56##    --       H  1 3                                   (14)                                                                              ##STR57##                                                                            H                                                                                 ##STR58##    --       H  0 4                                   (15)                                                                              ##STR59##                                                                            CH.sub.3                                                                         C.sub.6 H.sub.5 CH.sub.2                                                                    --       H  1 4                                   __________________________________________________________________________

The following examples describe the synthesis of R--A--S--Z.

EXAMPLE 87 Synthesis of N.sup.α -[3-(N.sup.α-benzoyl-tryptophyl)thioacetyl]-L-proline

A solution of 62 mg of N.sup.α -benzoyl-tryptophan in 0.5 ml redistilleddimethylformamide (DMF) is cooled in an ice-dry ice-acetone bath at -20°C. To this solution is added a cold solution of 35 mg of1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution is stirred at-10° C. for two hours and then added to a cold solution of 48 mg ofN-(2-mercaptoacetyl)-L-proline (from Example 17) in 1 ml of DMF which isneutralized with N-ethyl morpholine. The reaction mixture is stirred at-10° C. for an additional hour and then slowly warmed to roomtemperature. The solvent is removed under reduced pressure at 40° C. andethyl acetate is added to the residue. The mixture is cooled in an icebath and washed with 0.1N HCl and then three times with saturated NaClsolution. The solvent is removed with a rotary evaporator after dryingover anhydrous MgSO₄. The product is purified by liquid chromatographyon Sephadex LH- 20 using a 1.2 cm by 95 cm column and eluted withisopropanol. The peak fractions are pooled and the solvent removed underreduced pressure yielding the named product, as a foam-like material.

EXAMPLE 88 Synthesis of N.sup.α -([2-(N.sup.α-benzoylglycyl)thioethyl]sulfonyl)-L-proline

A solution of 33 mg of N.sup.α -benzoylglycine in 0.5 ml redistilleddimethylformamide (DMF) is cooled in an ice-dry ice-acetone bath at -20°C. To this solution is added a cold solution of 35 mg of1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution is stirred at-10° C. for two hours and then mixed with a cold solution of 48 mg ofN-[(2-mercaptoethyl)sulfonyl]-L-proline (from Example 28) in 1 ml of DMFwhich is neutralized with N-ethyl morpholine. The reaction mixture isstirred at -10° C. for an additional hour and then slowly warmed to roomtemperature. The solvent is removed under reduced pressure at 40° C. andethyl acetate is added to the residue. The mixture is cooled in an icebath and washed with 0.1N HCl and then three times with saturated NaClsolution. The solvent is removed with a rotary evaporator after dryingover anhydrous MgSO₄. The product is purified by liquid chromatographyon Sephadex LH-20 using a 1.2 cm by 95 cm column and elute withTHF:isopropanol, 3:7 (parts by volume). The peak fractions are pooledand the solvent removed under reduced pressure yielding the nameproduct.

EXAMPLE 89 Synthesis of N.sup.α -]3-(N.sup.α-tert-butyloxycarbonyl-phenylalanylthio)-2-trifluoromethylpropanoyl]-L-proline

A solution of 133 mg of N.sup.α -tert-butyloxycarbonylphenylalanine(N.sup.α -Boc-phenylalanine) in 0.5 ml redistilled dimethylformamide(DMF) is cooled in an ice-dry ice-acetone bath at -20° C. To thissolution is added a cold solution of 87 mg of 1,1'-carbonyldiimidazolein 1.0 ml of DMF. The solution is stirred at -10° C. for two hours andthen mixed with a cold solution of 119.5 mg ofN-(3-mercapto-2-trifluoromethyl)propanoyl-L-proline (from Example 33) in1 ml of DMF which is neutralized with N-ethyl morpholine. The reactionmixture is stirred at -10° C. for an additional hour and then slowlywarmed to room temperature. The solvent is removed under reducedpressure at 40° C. and ethyl acetate is added to the residue. Themixture is cooled in an ice and washed with 0.1N HCl and then threetimes with saturated NaCl solution. The solvent is removed with a rotaryevaporator after drying over anhydrous MgSO₄. The product is purified byliquid chromatography on Sephadex G-10 using a 1.2 cm by 95 cm columnand eluted with THF:isopropanol, 3:7 (parts by volume). The peakfractions are pooled and the solvent removed under reduced pressureyielding the named product.

EXAMPLE 90 Synthesis of N.sup.α-(3-phenylalanylthio-2-trifluoromethylpropanoyl)-L-proline

The product from Example 89 is deprotected by stirring a mixture of 30mg of the product, 50 μl of anisole and 200 μl of anhydroustrifluoroacetic acid (TFA) at room temperature for one hour. Anisole andTFA are removed under reduced pressure at 35° C. and the residue istriturated with anhydrous ether. The white residue is purified by liquidchromatography on Sephadex G-10 using a 1.2 cm by 95 cm column andeluted with 5% acetic acid. The peak fractions are pooled andfreeze-dried yielding the named compound.

EXAMPLE 91 Synthesis of N.sup.α -([3-(N.sup.α-acetyl-tyrosylthio)-1-oxopropyl]amino)-L-proline

A solution of 41.5 mg of N.sup.α -acetyl-tyrosine in 0.5 ml redistilleddimethylformamide (DMF) is cooled in an ice-dry ice-acetone bath at -20°C. To this solution is added a cold solution of 35 mg of1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution is stirred at-10° C. for two hours and then is added to a cold solution of 48 mg ofN-[(3-mercapto-1-oxopropyl)amino]-L-proline (from Example 39) in 1 ml ofDMF which is neutralized with N-ethyl morpholine. The reaction mixtureis stirred at -10° C. for an additional hour and then slowly warmed toroom temperature. The solvent is removed under reduced pressure at 40°C. and ethyl acetate is added to the residue. The mixture is cooled inan ice bath and washed with 0.1N HCL and then three times with saturatedNaCl solution. The organic solvent is removed with a rotary evaporatorafter drying over anhydrous MgSO₄. The product is purified by SephadexLH-20 column chromatography using a 1.2 cm by 95 cm column and elutedwith isopropanol. The peak fractions are pooled and the solvent isremoved under reduced pressure yielding the named product.

EXAMPLE 92 Synthesis of N.sup.α -[3-(N.sup.α-cyclopentanecarbonyl-isoleucylthio)-2-ethylpropanoyl]-L-3,4-dehydroproline

A solution of 52.5 mg of N.sup.α -cyclopentanecarbonylisoleucine in 0.5ml redistilled dimethylformamide (DMF) is cooled in an ice-dryice-acetone bath at -20° C. To this solution is added a cold solution of34 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution isstirred at -10° C. for two hours and then mixed with a cold solution of45.6 mg of N-(2-ethyl-3-mercaptopropanoyl)-L-3,4-dehydroproline (fromExample 42) in 1 ml of DMF which is neutralized with N-ethyl morpholine.The reaction mixture is stirred at -10° C. for an additional hour andthen slowly warmed to room temperature. The solvent is removed underreduced pressure at 40° C. and ethyl acetate is added to the residue.The mixture is cooled and washed with 0.1N HCl and then three times withsaturated NaCl solution. The solvent is removed with a rotary evaporatorafter drying over anhydrous MgSO₄. The product is purified by LH-20column chromatography using a 1.2 cm by 95 cm column and eluted withisopropanol. The peak fractions are pooled and the solvent is removedunder reduced pressure yielding the named product.

EXAMPLE 93 Synthesis of ([3-(N.sup.α -cyclopentanecarbonyl-N.sup.ε-tert-butyloxycarbonyl-L-lysyl-histidyl)thiopropanoyl]amino)-2-methylpropanoicacid

A solution of 73.5 mg of N.sup.α -cyclopentanecarbonyl-N.sup.ε-tert-butyloxycarbonyl-L-lysyl-histidine in 0.5 ml redistilleddimethylformamide (DMF) is cooled in an ice-dry ice-acetone bath at -20°C. To this solution is added a cold solution of 26 mg of1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution is stirred at-10° C. for two hours and then mixed with a cold solution of 36 mg of2-[(3-mercaptopropanoyl)amino]-2-methylpropanoic acid (from Example 46)in 1 ml of DMF which is neutralized with N-ethyl morpholine. Thereaction mixture is stirred at -10° C. for an additional hour and thenslowly warmed to room temperature. The solvent is removed under reducedpressure at 40° C. and ethyl acetate is added to the residue. Themixture is cooled in an ice water bath and washed with 1N citric acidand then three times with saturated NaCl solution. The solvent isremoved with a rotary evaporator after drying over anhydrous MgSO.sub.4. The product is purified by LH-20 column chromatography using a 1.2 cmby 95 cm column and eluted with THF:isopropanol, 3:7 (parts by volume).The peak fractions are pooled and the solvent is removed under reducedpressure yielding the named product.

EXAMPLE 94 Synthesis of ([3-(N.sup.α-cyclopentanecarbonyl-L-lysylhistidyl)thiopropanoyl]amino)-2-methylpropanoicacid

The N.sup.ε -Boc group is removed from the lysine by stirring a mixtureof 30 mg of the product from Example 93 with 50 ul anisole and 200 ul ofanhydrous trifluoroacetic acid (TFA) at room temperature for one hour.Anisole and TFA are removed under reduced pressure at 35° C. and theresidue is triturated with anhydrous ether. The residue is purified byliquid chromatography on Sephadex G-10 using a 1.2 cm by 95 cm columnand eluted with 5% acetic acid. The peak fractions are pooled andfreeze-dried yielding the named product.

EXAMPLE 95 Preparation of N.sup.α-[3-(Pyro-L-glutamyl-valyl)thiopropanoyl]-L-alanine

A solution of 87 mg of 1,1'-carbonyldiimidazole in 1.0 ml DMF is addedto a solution of 139 mg of pyro-L-glutamylvaline in 0.5 ml DMF at -15°C. The reaction mixture is stirred at -10° C. for 1 hour, and then amixture of 119.5 mg of N-(3-mercaptopropanoyl)-L-alanine (from Example50) and 0.072 ml of N-ethyl morpholine in 1 ml. DMF is added. Thereaction mixture is stirred at -10° C. for an additional hour and thenis slowly warmed to room temperature. DMF is removed under reducedpressure with a rotary evaporator at 40° C. and then 7 ml ethyl acetateand 2 ml 1N citric acid are added. The organic phase is washed two timeswith 1N citric acid and two times with saturated NaCl. The organic phaseis dried with anhydrous MgSO₄ and then filtered. Solvent is removedusing a rotary evaporator. The residue is purified on Sephadex G-25(1.2×99 cm) partition column chromatography with n-butanol:aceticacid:H₂ O (4:1:5 by volume) yielding the named product.

EXAMPLE 96 Preparation of N.sup.α-([2-(L-lysyl-leucyl)thiomethyl]-5-aminopentanoyl)-L-proline

A solution of 87 mg of 1,1'-carbonyldiimidazole in 1.0 ml DMF is addedto a solution of 139 mg of bis-Boc-L-lysylleucine in 0.5 ml DMF at -15°C. The reaction mixture is stirred at -10° C. for 1 hour, and then amixture of 119.5 mg of N-(5-amino-2-mercaptomethylpentanoyl)-L-proline(from Example 67) and 0.072 ml of N-ethyl morpholine in 1 ml. DMF isadded. The reaction mixture is stirred at -10° C. for an additional hourand then is slowly warmed to room temperature. DMF is removed underreduced pressure with a rotary evaporator at 40° C. and then 7 ml ethylacetate and 2 ml 1N citric acid are added. The organic phase is washedtwo times with 1N citric acid and two times with saturated NaCl. Theorganic phase is dried with anhydrous MgSO₄ and then filtered. Solventis removed using a rotary evaporator. The residue is purified onSephadex G-25 (1.2×99 cm) partition column chromatography withn-butanol:acetic acid:H₂ O (4:1:5 by volume). The bis-boc protectinggroup is removed by treatment with trifluoroacetic acid in anisole assubstantially described in Example 90 to yield the named product.

EXAMPLE 97 Preparation of N.sup.α-[(3-[L-arginyl-alanyl]thio)-2-(methylthiomethyl)propanoyl]-L-proline

A solution of 87 mg of 1,1'-carbonyldiimidazole in 1.0 ml DMF is addedto a solution of 139 mg of tri-Adoc-L-arginyl-alanine in 0.5 ml DMF at-15° C. The reaction mixture is stirred at -10° C. for 1 hour, and thena mixture of 119.5 mg ofN-[3-mercapto-2-(methylthiomethylpropanoyl]-L-proline (from Example 74)and 0.072 ml of N-ethyl morpholine in 1 ml. DMF is added. The reactionmixture is stirred at -10° C. for an additional hour and then is slowlywarmed to room temperature. DMF is removed under reduced pressure with arotary evaporator at 40° C. and then 7 ml ethyl acetate and 2 ml 1Ncitric acid are added. The organic phase is washed two times with 1Ncitric acid and two times with saturated NaCl. The organic phase isdried with anhydrous MgSO₄ and then filtered. Solvent is removed using arotary evaporator. The residue is purified on Sphadex G-25 (1.2×99 cm)partition column chromatography with n-butanol:acetic acid:H₂ O (4:1:5by volume). The tri-Adoc protecting group is removed by treatment withtrifluoroacetic acid in anisole as substantially described in Example 90to yield the named product.

EXAMPLE 98 Synthesis of N.sup.α -[(3-[N.sup.α-pyro-L-glutamyl-L-lysyl-phenylalanyl]-thio)-2-(methoxycarbonylmethyl)propanoyl]-L-proline

A solution of 73.5 mg of N.sup.α -pyro-L-glutamyl-N.sup.ε-tert-butyloxycarbonyl-L-lysyl-phenylalanine in 0.5 ml redistilleddimethylformamide (DMF) is cooled in an ice-dry ice-acetone bath at -20°C. To this solution is added a cold solution of 26 mg of1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution is stirred at-10° C. for two hours and then mixed with a cold solution of 36 mg ofN-[3-mercapto-2-(methoxycarbonylmethyl)propanoyl]-L-proline (fromExample 82) in 1 ml of DMF which is neutralized with N-ethyl morpholine.The reaction mixture is stirred at -10° C. for an additional hour andthen slowly warmed to room temperature. The solvent is removed underreduced pressure at 40° C. and ethyl acetate is added to the residue.The mixture is cooled in an ice water bath and washed with 1N citricacid and then three times with saturated NaCl solution. The solvent isremoved with a rotary evaporator after drying over anhydrous MgSO₄. Theproduct is purified by LH-20 column chromatography using a 1.2 cm by 95cm column and eluted with THF:isopropanol, 3:7 (parts by volume). Thepeak fractions are pooled and the solvent is removed under reducedpressure yielding the product N.sup.α -[(3-[N.sup.α-pyro-L-glutamyl-N.sup.ε-tertbutyloxycarbonyl-L-lysyl-phenylalanyl]thio-2-(methoxycarbonylmethyl)propanoyl]-L-proline.The tert-butyloxycarbonyl protecting group is removed as described inExample 94 to yield the named product.

EXAMPLE 99

By substituting the appropriate starting materials from Example 1-86using appropriate blocking groups where necessary into Examples 87-98and substantially following the procedures of Examples 87-98, thefollowing thioester compounds, R--A--S--Z, as defined in the followingtable are obtained.

                  TABLE 11                                                        ______________________________________                                        R                    A        Z                                               ______________________________________                                        H                    Phe      Ex. 20                                          formyl               Ala      Ex. 22                                          L-arginyl            His      Ex. 25                                          pyro-L-glutamyl      Leu      Ex. 26 (2)                                      propanoyl            Phe      Ex. 26 (8)                                      cyclopentanecarbonyl Tyr      Ex. 26 (15)                                     formyl               Phe      Ex. 26 (21)                                     L-lysyl              Gly      Ex. 26 (29)                                     butanoyl             Trp      Ex. 29 (1)                                      phenylacetyl         Phe      Ex. 29 (5)                                      L-lysyl              Phe      Ex. 29 (9)                                      acetyl               Val      Ex. 34                                          pyro-L-glutamyl-L-lysyl                                                                            Ile      Ex. 35 (4)                                      acetyl               His      Ex. 35 (10)                                     t-butyloxycarbonyl   Phe      Ex. 35 (14)                                     benzoyl              Phe      Ex. 40 (1)                                      phenylpropanoyl      Gly      Ex. 40 (6)                                      cyclopentanecarbonyl-L-lysyl                                                                       Val      Ex. 40 (11)                                     H                    His      Ex. 40 (16)                                     benzoyl              Trp      Ex. 40 (19)                                     H                    Tyr      Ex. 43                                          t-butyloxycarbonyl   Leu      Ex. 44 (3)                                      L-lysyl              Phe      Ex. 44 (7)                                      pyro-L-glutamyl-L-lysyl                                                                            Gly      Ex. 44 (11)                                     t-butyloxycarbonyl   Trp      Ex. 47                                          benzoyl              Ala      Ex. 48 (2)                                      benzoyl              Phe      Ex. 48 (9)                                      H                    Ile      Ex. 48 (13)                                     cyclopentanecaronyl  His      Ex. 51                                          pyro-L-glutamyl      Gly      Ex. 52 (1)                                      phenylacetyl         Ala      Ex. 52 (7)                                      benzoyl              Val      Ex. 55                                          L-arginyl            Trp      Ex. 57                                          cyclopentanecarbonyl Phe      Ex. 68 (3)                                      formyl               His      Ex. 68 (11)                                     pyro-L-glutamyl      Tyr      Ex. 68 (15)                                     benzoyl              Ala      Ex. 68 (22)                                     propanoyl            Phe      Ex. 71                                          L-lysyl              Ile      Ex. 75                                          butanoyl             Val      Ex. 78 (2)                                      H                    Gly      Ex. 78 (8)                                      cyclopentanecarbonyl-L-lysyl                                                                       Phe      Ex. 78 (14)                                     phenylpropanoyl      Trp      Ex. 78 (18)                                     L-lysyl              Ile      Ex. 83                                          cyclopentanecarbonyl His      Ex. 85                                          benzoyl              Leu      Ex. 86 (1)                                      H                    Ala      Ex. 86 (5)                                      t-butyloxycarbonyl   Phe      Ex. 86 (11)                                     ______________________________________                                    

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A new compound having the general formula I.R--A--S--Z, and the physiologically acceptable salts thereof, wherein:Ris selected from hydrogen, formyl, acetyl, propanoyl, butanoyl,phenylacetyl, phenylpropanoyl, benzoyl, cyclopentanecarbonyl,tert-butyloxycarbonyl, cyclopentanecarbonylL-lysyl,pyro-L-glutamyl-L-lysyl, L-arginyl, L-lysyl and pyro-L-glutamyl, A isselected from phenylalanyl, glycyl, alanyl, tryptophyl, tyrosyl,isoleucyl, leucyl, histidyl and valyl and the -amino group of A is inamide linkage with R when R is an acyl group and forms a primary aminogroup with R when R is hydrogen; S is a sulfur atom joined to A in athioester linkage; and Z is selected from the following: ##STR60##wherein (i) R₁ and R₁ ' are each hydrogen or halogen, and R₂ and R₃ areeach hydrogen, lower alkyl or trifluoromethyl provided that only one ofR₂ and R₃ may be trifluoromethyl and further provided that at least oneof R₁, R₁ ', R₂ and R₃ must be halogen or trifluoromethyl; (ii) R₂₃ ishydrogen or lower alkyl and (iii) m is 0 or 1; ##STR61## wherein (i) R₄and R₅ are each hydrogen, lower alkyl or phenyl-lower alkylene; (ii) nis 1, 2 or 3; (iii) R₆ is hydrogen or hydroxy or when n is 2, R₆ mayalso be halogen, and (iv) m and R₂₃ are as stated in II. above;##STR62## wherein (i) R₇ is hydrogen, lower alkanoyl or amino(imino)-methyl; (ii) p is 0 or an integer of from 1 to 4; (iii) R₂₅ ishydrogen but when m is 1, p is 0, R₄ is hydrogen and R₇ is loweralkanoyl, R₂₅ may also be lower alkyl; (iv) R₈ is selected fromhydrogen, lower alkyl and hydroxy lower alkylene when R₉ is selectedfrom hydrogen, lower alkyl, phenyl, phenyl-lower alkylene, hydroxy-loweralkylene, hydroxyphenyl-lower alkylene, amino-lower alkylene,guanidino-lower alkylene, mercapto-lower alkylene, loweralkyl-thio-lower alkylene, imidazolyl-lower alkylene, indolyl-loweralkylene, carbamoyl-lower alkylene and carboxy-lower alkylene but R₈ andR₉ may together constitute a (CH₂)_(v) bridge wherein v is 3 or 4, thusforming a 5 or 6-membered ring with the N and C to which R₈ and R₉ arerespectively attached and in such instance when v is 3 one hydrogen of(CH₂)_(v) may be replaced by OH or halogen and when v is 4, one suchhydrogen may be replaced by OH; (v) m and R₂₃ are each as defined in II.above; and (iv) R₄ is as defined in III. above, provided further,however, that m and p may not both be
 0. ##STR63## wherein (i) r₁₀ ishydrogen or lower alkyl; (ii) R₁₁ is hydrogen, lower alkyl or loweralkanoyl; (iii) X is 0 or S; (iv) m and R₂₃ are as stated in II. above,and (v) R₈, R₉ and p are as stated in IV. above; ##STR64## wherein (i)R₁₂ is selected from carboxy, lower alkoxycarbonyl, carbamoyl,N-substituted carbamoyl and cyano; (ii) m and R₂₃ are as stated in II.above; and (iii) R₈, R₉ and p are as stated in IV. above; ##STR65##wherein (i) R₁₃ is hydrogen, lower alkyl or phenyl-lower alkylene; (ii)R₁₄ is selected from hydrogen, lower alkyl, phenyl-lower alkylene,hydroxy-lower alkylene, amino-lower alkylene, guanidino-lower alkylene,imidazolyl-lower alkylene, indolyl-lower alkylene, mercapto-loweralkylene, lower alkyl-thio-lower alkylene, carbamoyl-lower alkylene andcarboxy-lower alkylene; (iii) R₄ and R₅ are as stated in III. above; and(iv) q is 0, 1 or 2 ##STR66## wherein (i) R₁₅ and R₁₆ are each hydrogen,lower alkyl, phenyl or phenyl-lower alkylene; (ii) r is 0, 1 or 2; (iii)s is 1, 2, or 3; (iv) R₁₇ is hydrogen, hydroxy or lower alkyl and when sis 2, R₁₇ may also be halogen; and (v) R₂₄ is hydroxy, amino or loweralkoxy; provided that when R₂₄ is OH, s is 2, and R₁₇ is H or OH:(a) ifr is 0, R₁₆ may not be methyl or H, and (b) if r is 1, R₁₅ and R₁₆ maynot both be H and R₁₆ may not be methyl if R₁₅ is H ##STR67## wherein(i) R₁₈ is hydrogen or lower alkyl; (ii) R₁₉ and R₂₀ are each loweralkyl and may together constitute a (CH₂)_(w) bridge wherein w is 4, toform a ring of 5-carbons with the carbon to which they are eachattached; (iii) R₂₁ is hydrogen or lower alkyl and may constitute withR₁₉ a (CH₂)_(x) bridge wherein x is 3, to form a five-membered ring withthe N and C to which they are respectively attached;provided, however,that when R₁₉ and R₂₁ constitute (CH₂)_(x), R₁₈ may not be hydrogen ormethyl ##STR68## wherein (i) R₂₂ is hydrogen or lower alkyl; (ii) t andu are each 0 or 1; (iii) R₂₃ is as stated in II. above;provided,however, that u may not be zero when R₂₃ is H; and ##STR69## wherein (i)z is 2 or 3; (ii) R₁₀ is as stated in V. above, and (iii) R₂₃ is asstated in II above.
 2. The compound of claim 1 wherein A is glycyl,phenylalanyl or tryptophyl.
 3. The compound of claim 1 wherein R ishydrogen, acetyl, benzoyl, cyclopentanecarbonyl, tert-butyloxycarbonyl,cyclopentanecarbonyl-L-lysyl or pyro-L-glutamyl.
 4. The compound ofclaim 1 wherein Z is defined by formula II.
 5. The compound of claim 1wherein Z is defined by formula III.
 6. The compound of claim 1 whereinZ is defined by formula IV.
 7. The compound of claim 1 wherein Z isdefined by formula V.
 8. The compound of claim 1 wherein Z is defined byformula VI.
 9. The compound of claim 1 wherein Z is defined by formulaVII.
 10. The compound of claim 1 wherein Z is defined by formula VIII.11. The compound of claim 1 wherein Z is defined by formula IX.
 12. Thecompound of claim 1 wherein Z is defined by formula X.
 13. The compoundof claim 1 wherein Z is defined by formula XI.
 14. The compound of claim5 wherein n is
 2. 15. The compound of claim 6, 7 or 8 wherein R₈ and R₉together form a --CH₂ CH₂ CH₂ -- bridge which completes a ring of 5atoms with the nitrogen and carbon to which they are attached, onecarbon optionally bearing a hydroxy group or a halogen group.
 16. Thecompound of claim 10 wherein s is
 2. 17. The compound of claim 11wherein R₁₉ and R₂₁ together form a --CH₂ CH₂ CH₂ -- bridge whichcompletes a ring of 5 atoms with the nitrogen and carbon to which theyare attached.
 18. The compound of claim 12 wherein u is
 0. 19. Thecompound of claim 13 wherein z is
 2. 20. The compound of claim 1, 4 or 9wherein R is benzoyl and A is phenylalanyl.
 21. The compound of claim 5or 14 wherein R is benzoyl and A is phenylalanyl.
 22. The compound ofclaim 6, 7, 8 or 15 wherein R is benzoyl and A is phenylalanyl.
 23. Thecompound of claim 10 or 16 wherein R is benzoyl and A is phenylalanyl.24. The compound of claim 11 or 17 wherein R is benzoyl and A isphenylalanyl.
 25. The compound of claim 12 or 18 wherein R is benzoyland A is phenylalanyl.
 26. The compound of claim 13 or 19 wherein R isbenzoyl and A is phenylalanyl.
 27. A method for inhibiting angiotensinconverting enzyme in vivo which comprises administering to a mammal inneed of such treatment an effective dose of the compound of claim
 1. 28.A method for inhibiting angiotensin converting enzyme in vivo whichcomprises administering to a mammal in need of such treatment aneffective dose of the compound of claim
 2. 29. A method for inhibitingangiotensin converting enzyme in vivo which comprises administering to amammal in need of such treatment an effective dose of the compound ofclaim
 3. 30. A method for inhibiting angiotensin converting enzyme invivo which comprises administering to a mammal in need of such treatmentan effective dose of the compound of claim
 4. 31. A method forinhibiting angiotensin converting enzyme in vivo which comprisesadministering to a mammal in need of such treatment an effective dose ofthe compound of claim
 5. 32. A method for inhibiting angiotensinconverting enzyme in vivo which comprises administering to a mammal inneed of such treatment an effective dose of the compound of claim
 6. 33.A method for inhibiting angiotensin converting enzyme in vivo whichcomprises administering to a mammal in need of such treatment aneffective dose of the compound of claim
 7. 34. A method for inhibitingangiotensin converting enzyme in vivo which comprises administering to amammal in need of such treatment an effective dose of the compound ofclaim
 8. 35. A method for inhibiting angiotensin converting enzyme invivo which comprises administering to a mammal in need of such treatmentan effective dose of the compound of claim
 9. 36. A method forinhibiting angiotensin converting enzyme in vivo which comprisesadministering to a mammal in need of such treatment an effective dose ofthe compound of claim
 10. 37. A method for inhibiting angiotensinconverting enzyme in vivo which comprises administering to a mammal inneed of such treatment an effective dose of the compound of claim 11.38. A method for inhibiting angiotensin converting enzyme in vivo whichcomprises administering to a mammal in need of such treatment aneffective dose of the compound of claim
 12. 39. A method for inhibitingangiotensin converting enzyme in vivo which comprises administering to amammal in need of such treatment an effective dose of the compound ofclaim
 13. 40. A method for inhibiting angiotensin converting enzyme invivo which comprises administering to a mammal in need of such treatmentan effective dose of the compound of claim
 14. 41. A method forinhibiting angiotensin converting enzyme in vivo which comprisesadministering to a mammal in need of such treatment an effective dose ofthe compound of claim
 15. 42. A method for inhibiting angiotensinconverting enzyme in vivo which comprises administering to a mammal inneed of such treatment an effective dose of the compound of claim 16.43. A method for inhibiting angiotensin converting enzyme in vivo whichcomprises administering to a mammal in need of such treatment aneffective dose of the compound of claim
 17. 44. A method for inhibitingangiotensin converting enzyme in vivo which comprises administering to amammal in need of such treatment an effective dose of the compound ofclaim
 18. 45. A method for inhibiting angiotensin converting enzyme invivo which comprises administering to a mammal in need of such treatmentan effective dose of the compound of claim
 19. 46. A method forinhibiting angiotensin converting enzyme in vivo which comprisesadministering to a mammal in need of such treatment an effective dose ofthe compound of claim
 20. 47. A method for inhibiting angiotensinconverting enzyme in vivo which comprises administering to a mammal inneed of such treatment an effective dose of the compound of claim 21.48. A method for inhibiting angiotensin converting enzyme in vivo whichcomprises administering to a mammal in need of such treatment aneffective dose of the compound of claim
 22. 49. A method for inhibitingangiotensin converting enzyme in vivo which comprises administering to amammal in need of such treatment an effective dose of the compound ofclaim
 23. 50. A method for inhibiting angiotensin converting enzyme invivo which comprises administering to a mammal in need of such treatmentan effective dose of the compound of claim
 24. 51. A method forinhibiting angiotensin converting enzyme in vivo which comprisesadministering to a mammal in need of such treatment an effective dose ofthe compound of claim
 25. 52. A method for inhibiting angiotensinconverting enzyme in vivo which comprises administering to a mammal inneed of such treatment an effective dose of the compound of claim 26.53. A method for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 1. 54. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 2. 55. A methodfor reducing in vivo the blood pressure of a mammal in the hypertensivestate which comprises administering an effective dose of the compound ofclaim
 3. 56. A method for reducing in vivo the blood pressure of amammal in the hypertensive state which comprises administering aneffective dose of the compound of claim
 4. 57. A method for reducing invivo the blood pressure of a mammal in the hypertensive state whichcomprises administering an effective dose of the compound of claim 5.58. A method for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 6. 59. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 7. 60. A methodfor reducing in vivo the blood pressure of a mammal in the hypertensivestate which comprises administering an effective dose of the compound ofclaim
 8. 61. A method for reducing in vivo the blood pressure of amammal in the hypertensive state which comprises administering aneffective dose of the compound of claim
 9. 62. A method for reducing invivo the blood pressure of a mammal in the hypertensive state whichcomprises administering an effective dose of the compound of claim 10.63. A method for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 11. 64. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 12. 65. Amethod for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 13. 66. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 14. 67. Amethod for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 15. 68. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 16. 69. Amethod for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 17. 70. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 18. 71. Amethod for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 19. 72. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 20. 73. Amethod for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 21. 74. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 22. 75. Amethod for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 23. 76. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim
 24. 77. Amethod for reducing in vivo the blood pressure of a mammal in thehypertensive state which comprises administering an effective dose ofthe compound of claim
 25. 78. A method for reducing in vivo the bloodpressure of a mammal in the hypertensive state which comprisesadministering an effective dose of the compound of claim 26.